Phage displaying system expressing single chain antibody

ABSTRACT

Disclosed are nucleic acid libraries for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and for facilitating production of a disulfide-stabilized single chain antibody. Also disclosed are host cell libraries and phage libraries including the nucleic acid libraries. Further disclosed are methods for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and methods for producing a disulfide-stabilized single chain antibody and non-fusion form thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/897,850, filed on May 20, 2013, which is a divisional of U.S.application Ser. No. 12/854,632, filed on Aug. 11, 2010, which claimspriority to U.S. Provisional Application No. 61/232,819, filed on Aug.11, 2009. The contents of all prior applications are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention is related to a phage displaying system expressingdisulfide-stabilized single chain antibody variable fragments (sc-dsFv).

BACKGROUND OF THE INVENTION

A single chain variable fragment (scFv) is a single polypeptide chainantibody fragment having a light chain variable domain and a heavy chainvariable domain, with a flexible linkage peptide connecting the twodomains. An scFv displayed as a fusion protein N-terminal to the pIIIminor capsid protein on filamentous phage surface is one of the mostprominent methods in antibody engineering. It was reported that thesmall size of the scFv enabled superior tissue-penetrating capabilitiesover whole IgG or Fab fragment, making scFv an ideal scaffold fordesigning tumor-homing molecules carrying therapeutic or imaging agents(Michnick, S. W., and Sidhu, S. S. (2008) Nat Chem Biol 4(6), 326-329).

Yet, under practical application conditions, an scFv scaffold tends toform aggregation. The aggregation has much to do with the stability ofthe two variable domains and the dimeric interface. The instability ofthe scFv structure also compromises the fidelity in reproducing theantibody gene products on phage surface, causing biases in favor of morestable scFv molecules over the less stable ones, or selecting non-foldedstructures on phage surfaces but nevertheless binding to an antigen.This structural instability thus impacts negatively on the applicationsof scFv in biotechnology and medical uses.

One way to stabilize the scFv scaffold is to engineer a disulfide bondbetween the two Fv domains, so that the variable domains can becovalently linked with a disulfide bond. Single chaindisulfide-stabilized Fv fragment (sc-dsFv) format was constructed in asingle polypeptide chain, as in scFv, with a disulfide framework region(Young, N. M. et al., (1995) FEBS Lett 377(2), 135-139; Worn, A., andPluckthun, A. (1999) Biochemistry 38(27), 8739-8750). The sc-dsFvmolecules could be expressed in E. coli, but not be expressed on phagesurface or as soluble form secreted by E. coli in a culture medium,mostly due to severely decreased yield because of the introduction ofinterface cysteines (Worn, A., and Pluckthun, A. (2001) J Mol Biol305(5), 989-1010).

Up to now, phage-displayed sc-dsFv libraries and their applications havenot been established.

BRIEF SUMMARY OF THE INVENTION

The invention provides a methodology to systematically optimize thesignal sequences for phage-displayed protein expression, for which theexpression with conventional signal sequences was not viable. Theoptimized signal sequences and related discovering methodologies led tothe establishment of phage display systems with the sc-dsFv format,enabling the demonstration and comparison of the performance of thesc-dsFv phage display platform with that of the conventional scFvplatform.

Accordingly, in one aspect, the present invention provides a nucleicacid library for identifying a signal peptide that facilitatesproduction of disulfide-stabilized single chain antibody. The libraryincludes a plurality of expression constructs, each of which includes: afirst nucleotide sequence encoding a signal peptide, and a secondnucleotide sequence encoding a single chain antibody capable of formingan interface disulfide bond. The second nucleotide sequence is located3′ downstream to the first nucleotide. The signal peptide has the aminoacid sequence of:

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b) (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c) (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH.Each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurringamino acid residues.

In another aspect, the invention provides a host cell library foridentifying a signal peptide that facilitates production ofdisulfide-stabilized single chain antibody. The library includes aplurality of host cells each containing an expression construct thatincludes: a first nucleotide sequence encoding a signal peptide, and asecond nucleotide sequence encoding a single chain antibody capable offorming an interface disulfide bond; the second nucleotide sequence islocated 3′ downstream to the first nucleotide; the signal peptide hasthe amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b) (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c) (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH.each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurringamino acid residues.

In another aspect, the invention provides a phage library foridentifying a signal peptide that facilitates production ofdisulfide-stabilized single chain antibody. The library has a pluralityof phage particles each containing a disulfide-stabilized single chainantibody fused with a coat protein on the surface of the phage. Thephage library is prepared by the steps of: providing a host cellcontaining an expression construct, and culturing the host cell in amedium under conditions allowing expression of the plurality of phageparticles; the expression construct that includes a first nucleotidesequence encoding a signal peptide, a second nucleotide sequenceencoding a single chain antibody capable of forming an interfacedisulfide bond, the second nucleotide sequence being located 3′downstream to the first nucleotide, and a third nucleotide sequenceencoding a phage envelop protein; the third nucleotide sequence beinglocated 3′ downstream to the second nucleotide sequence; the signalpeptide has the amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b) (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c) (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH,each of X₁-X₁₀ in (a), (b), and (c) being one of the 20 naturallyoccurring amino acid residues.

In addition, the invention provides a sc-dsFv phage display platform.According to the invention, a large scale screening for optimal signalsequences was carried out. In one example of the invention, the signalsequences that were effective for phage-displayed sc-dsFv and non-fusionsoluble sc-dsFv secretion in E. coli Amber suppressor strain ER2738 werescreened to obtain the sequence preference patterns emerged from theoptimum signal sequences.

In still another aspect, the present invention provides an isolatednucleic acid, having a first nucleotide sequence encoding a signalpeptide, and a second nucleotide sequence encoding a single chainantibody capable of forming an interface disulfide bond. The signalpeptide has the amino acid sequence of

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in whichX₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I,L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W;X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q,R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y;(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in whichX₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T,V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, orY; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A,C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉ is A, C, D, F, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M,P, Q, R, S, or T; or(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in whichX₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D,F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M,N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

In a further aspect, the present invention provides a host cellcontaining the nucleic acid described above.

In a further more aspect, the present invention provides a phagecontaining a disulfide-stabilized single chain antibody fused with itscoat protein on the surface. The phage is prepared by a method havingthe steps of: providing the above-described host cell, and culturing thehost cell in a medium under conditions allowing expression of the phage.

In further another aspect, the present invention provides a method forproducing a disulfide-stabilized single chain antibody. The methodincludes the steps of providing a host cell containing an expressionconstruct, and culturing the host cell in a medium under conditionsallowing expression of the disulfide-stabilized single chain antibody.The expression construct includes a first nucleotide sequence encoding asignal peptide, and a second nucleotide sequence encoding a single chainantibody capable of forming an interface disulfide bond; the signalpeptide has the amino acid sequence of:

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in whichX₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I,L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W;X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q,R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y;(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in whichX₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T,V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, orY; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A,C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X₉ is A, C, D, F, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M,P, Q, R, S, or T; or(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in whichX₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D,F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M,N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

In addition, the present invention provides a new signal peptide thatfacilitates production of disulfide-stabilized single chain antibody,and the nucleic acid encoding the signal peptide.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood, however, that theinvention is not limited to the precise arrangements andinstrumentalities shown.

FIG. 1 is a schema showing the signal sequence in pCANTAB5E and theconstructs of DNA libraries to diversify the tentative signal sequenceresponsible for the expression of the phage-displayed pIII fusionproteins.

FIG. 2 is a set of diagrams showing the results of the increased bindingto VEGF for phage-displayed sc-dsFv signal sequence variants enrichedfrom the three libraries after selection/amplification cycles, including(A) after each round of selection/amplification cycle, the values of thebinding of the rescued phage to immobilized VEGF as measured with ELISA.The ELISA signal strengths are shown in the y-axis, as functions of theselection/amplification cycle; and (B) the numbers of output phageparticles titered after each round of selection/amplification cycle foreach of the three libraries; the output phage titers, as shown in they-axis, were plotted against the number of the selection/amplificationcycles.

FIG. 3 is a schema showing the DNA construct of the S5 anti-VEGF sc-dsFvas a pIII fusion protein in the pCANTAB5E phagemid.

FIG. 4 is a diagram showing VEGF-binding strengths of thephage-displayed anti-VEGF sc-dsFv's from various signal sequencevariants with or without fXa digestion. Eight variants with maximal fXadigestion resistance from a 96-well ELISA plate containing 96 randomlypicked variants were selected from each of the VEGF-binding enrichedlibraries after the 4^(th) round of selection/amplification cycle. Thesevariants were cultured and the rescued phages were allowed to bind toimmobilized VEGF with (grey histogram) and without (black histogram) thefXa treatment, and the VEGF-binding strengths (y-axis) were measuredwith ELISA. The error bars were derived from three repeats of the ELISAmeasurement.

FIG. 5 is a diagram showing the binding strengths of phage-displayedanti-HAs scFv/sc-dsFv. One of the scFv phages with specific bindingability to H5, 8a, and the other one with broad-spectrum ability to HAs,12a, were engineered to disulfide-stabilized scFv (ds-scFv) formats; thesc-dsFv construct was different from the scFv construct in the mutations(L:Gly100Cys & H:Gly44Cys). Av1 was negative control of an scFvdisplayed on the phage; and TAA means the phage does not contain anydisplayed protein; and various HA subtypes were precoated to ELISA wellsto determined binding activity, and the error bars were derived fromthree repeats of the ELISA measurements.

FIG. 6 is a set of diagrams showing correlations between sc-dsFv foldingquality and resistance to fXa digestion. (A) shows a comparison of theextents (percentages) of the interface disulfide bond formation of thesc-dsFv from the optimum signal sequence variants from L4; both of theaxes show the ratio (percent) of the ELISA signal for VEGF-binding afterthe fXa treatment over the ELISA signal for VEGF-binding before the fXatreatment; the y-axis shows the data from secreted sc-dsFv; the x-axisshows the data from phage-displayed sc-dsFv. (B) shows a comparison ofthe extents (percentages) of the interface disulfide bond formation(y-axis) with the folding quality (x-axis) of the sc-dsFv from theoptimum signal sequence variants from L4. The sc-dsFv folding quality(x-axis) is represented as the sc-dsFv-VEGF binding ELISA signal dividedby western blot signal probed with anti-E tag antibody (E/W, VEGFbinding signal divided by secreted sc-dsFv quantity), and then the ratiois normalized with that of anti VEGF scFv (fXa+) (CE/CW, VEGF bindingsignal divided by secreted scFv quantity), that is, the folding qualityis quantified with the ratio: (E/W)/(CE/CW); the error bars in each datapoint indicate the standard deviations from three repeats of theexperiment; the coefficient of determination R2 and the p-value fromSpearman's rank correlation coefficient was shown in each panel.

FIG. 7 is a set of diagrams showing stability test of soluble sc-dsFv;including (A) showing the results of the soluble sc-dsFv incubated at37° C. as the indicated time shown in the x-axis, and the bindingcapacities estimated with ELISA against VEGF, shown in y-axis; the ELISAsignal was normalized against that of the secreted protein kept at 4°C.; and (B) showing the fXa resistance percentages of the solublesc-dsFv plotted against the end binding capacities after 12 days ofincubation in 37° C.; the error bars in each data point indicate thestandard deviations from three repeats of the experiment, and thecoefficient of determination R2 and the p-value from Spearman's rankcorrelation coefficient are shown in the panel.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The articles “a” and “an” are used herein to refer to one or more thanone (i.e., at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

Amino acids can be expressed by three letters or one letters. Table 1lists standard amino acid abbreviations.

TABLE 1 Standard amino acid abbreviations Amino Acid 3-Letter 1-LetterAlanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp DCysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

Very little is known as to why some sc-dsFv constructs could not beexpressed on phage surface, and why the disulfide bonds of the newlysynthesized preprotein can only be formed in the oxidizing environmentof periplasm. The mechanism for the translocation of the nascentunfolded polypeptide chain from the translation site in the cytoplasmacross the periplasmic membrane could be a key determinant for thefolding. It was unexpectedly found in the invention that for theexpression of the displayed protein on the phage surface, alternativesequences in the signal peptide region can modulate the expression leveland folding quality of the displayed protein. Accordingly, the inventionprovides a methodology to systematically optimize the signal sequencesfor phage-displayed protein expression. Based on the optimized signalsequences and the methodologies of the invention, phage display systemswith the sc-dsFv format are established.

According to the present invention, a nucleic acid library foridentifying a signal peptide that facilitates production ofdisulfide-stabilized single chain antibody is provided. The library hasa plurality of expression constructs, each of which includes: a firstnucleotide sequence encoding a signal peptide, and a second nucleotidesequence encoding a single chain antibody capable of forming aninterface disulfide bond. The second nucleotide sequence is located 3′downstream to the first nucleotide. The signal peptide has the aminoacid sequence of:

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b) (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c) (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH,each of X₁-X₁₀ in (a), (b), and (c) being one of the 20 naturallyoccurring amino acid residues.

In one embodiment, each of the expression constructs further includes athird nucleotide encoding a phage coat protein, and the third nucleotidesequence being located 3′ downstream to the second nucleotide.

The term “signal peptide” or “signal sequence” used herein refers to ashort (i.e. 3-60 amino acids long) peptide chain that directs thetransport of a protein. The signal peptide is known to be responsiblefor the sec system-dependent translocation of the sc-dsFv-pIII fusionfrom the translation site in cytoplasm across the periplasmic membrane,a critical process for the integration of the displayed protein on therecombinant phage. Considering the vast signal peptide sequence spaceneeded to be explored, the present invention provides biologicalcombinatorial strategies to diversify the signal peptide sequences withsynthetic phage display libraries. The variants in the phage librarieswere selected and screened for high expression capabilities, so as toidentify the key regions of the signal peptide sequences, including theoptimal amino acid sequences, positions and types that are effectivelyresponsible for the sc-dsFv expression on phage surface.

The term “single chain variable fragment” or “scFv” used herein refersto a single polypeptide chain antibody fragment construct encoding afirst variable region and a second variable region, with a flexiblelinkage peptide connecting the two domains. The first and the secondvariable region can be either a light chain or a heavy chain variableregion. The recombinant antibody fragment frequently retainsantigen-recognizing capability rivaling that of the parent antibody. Oneshortcoming of the scFv scaffold is the aggregation tendency of the scFvmolecules under physiological and storage conditions. The aggregationmechanism has much to do with the stability of the two variable domainsand the dimeric interface. This structural instability has thus impactednegatively on the utilities of scFv, leading to uncertainties to theoutcomes of the selected and screened scFv molecules in terms of theirpotential applications in biomedicine.

The term “disulfide-stabilized single chain antibody variable fragment”or “sc-dsFv” used herein refers to a single polypeptide chain containingtwo variable regions capable of forming an interface disulfide bond,where each of the two variable regions may be a heavy chain variableregion or a light chain variable region. According to the invention, thesc-dsFv-pIII fusion protein can be prepared by using the optimal signalsequences capable of directing the sc-dsFv expression on phage surface.

In an embodiment of the invention, the overlapping segments encompassingthe complete signal sequence region governing the protein trafficking ofthe model anti-VEGF sc-dsFv fusion protein were searched with biologicalcombinatorial methodology for sequence preferences leading to effectiveexpression of the sc-dsFv. The engineering platform established for thedisulfide-stabilized antibody variable domain fragment as demonstratedcould be used to prepare many of scFv molecules in a more stablestructure, which could be carried out under harsh conditions, and havelonger shelf-life.

According to one embodiment of the invention, to select signal sequencesfor effective expression of anti-VEGF sc-dsFv on M13 phage surface,phage display libraries L2, L3 and L4 were constructed to diversify thesignal sequence as shown in FIG. 1, where M13pIII-pelB indicated thesignal sequence being the wild type signal sequence for pIII in M13phage genome in connection with pelB peptidase cleavage site. Thecomplexities of the L2, L3 and L4 phage display library were 3.1×10⁹,3.7×10⁹, and 1.5×10⁹, respectively. These libraries were designed toefficiently diversify the signal peptide sequences on identifying theoptimum signal peptides for expression sc-dsFv.

In one example of the invention, the expression construct is a phagemid.Among the expression constructs, the nucleotide sequence of the signalpeptide, sc-dsFv and the phage coat protein could be operatively linkedin a random order. In one preferred example of the invention, the secondnucleotide sequence encoding sc-dsFv is located 3′ downstream to thefirst nucleotide encoding the signal peptide, and the third nucleotidesequence encoding the phage coat protein is located 3′ downstream to thesecond nucleotide sequence.

In one embodiment of the invention, a sc-dsFv library, containing morethan one billion sc-dsFv variants, is propagated with an E. coli vectorof bacterial phage origin following the method as described byMcCafferty, J. et al. (Nature 348(6301), 552-554, 1990). The recombinantphages displaying the sc-dsFv variants can be selected or screened forantigen-binding and re-amplified with the host cells, i.e. E. coli.

Furthermore, the present invention provides a host cell library foridentifying a signal peptide that facilitates production ofdisulfide-stabilized single chain antibody. The library includes aplurality of host cells each containing the aforementioned expressionconstructs.

The present invention also provides a phage library for identifying asignal peptide that facilitates production of disulfide-stabilizedsingle chain antibody. The library includes a plurality of phageparticles each containing a disulfide-stabilized single chain antibodyfused with a coat protein on the surface of said phage. The phagelibrary is prepared by the steps of: providing a host cell containing anexpression construct, and culturing the host cell in a medium underconditions allowing expression of the plurality of phage particles. Theexpression construct includes (1) a first nucleotide sequence encoding asignal peptide, (2) a second nucleotide sequence encoding a single chainantibody capable of forming an interface disulfide bond, the secondnucleotide sequence being located 3′ downstream to the first nucleotide,and (3) a third nucleotide sequence encoding a phage envelop protein,the third nucleotide sequence being located 3′ downstream to the secondnucleotide sequence. The signal peptide has the amino acid sequence of

(a)  (SEQ ID NO: 1) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH, (b) (SEQ ID NO: 2) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH,  or (c) (SEQ ID NO: 3) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH,each of X₁-X₁₀ in (a), (b), and (c) is one of the 20 naturally occurringamino acid residues.

On the other hand, a sc-dsFv engineering platform is established forpreparation of scFv molecules in a more stable structure in the presentinvention. Accordingly, the present invention provides an isolatednucleic acid that has a first nucleotide sequence encoding a signalpeptide, and a second nucleotide sequence encoding a single chainantibody capable of forming an interface disulfide bond. The secondnucleotide sequence is located 3′ downstream to the first nucleotide.The signal peptide has the amino acid sequence of

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in whichX₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I,L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W;X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q,R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y;(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in whichX₁ is A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D,F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I,L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S,T, V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W,or Y; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ isA, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉ is A, C, D, F, H, I,L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L,M, P, Q, R, S, or T; or(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in whichX₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D,F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M,N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

According to the invention, the nucleic acid further includes a thirdnucleotide encoding a phage coat protein. The third nucleotide sequenceis located 3′ downstream to the second nucleotide sequence.

In one example of the invention, anti-VEGF sc-dsFv phage displayplatform was developed. As shown in FIG. 1, expression constructs foridentifying a signal peptide that facilitates production ofdisulfide-stabilized single chain antibody were designed. Each of thethree DNA libraries (L2, L3, and L4) contained ten consecutive NNKdegenerate codons covering overlapping regions around the signalsequence. N stands for A, G, T, or C, 25% each; K stands for G or T, 50%each. The NNK degenerated codon represents 32 possible tripletcombinations, encoding all 20 natural amino acids and an amber stopcodon (TAG). Each of the phage display libraries was selected forbinding against immobilized VEGF. The trends of enrichment of theVEGF-binding phage variants from each of the three libraries, plotted asfunctions of the number of selection/amplification cycle, are shown inFIG. 2. The enrichment trends were similar among the variants from thethree libraries. This result indicates that the signal sequence regionscovered by the three signal sequence libraries (see FIG. 1) can all beoptimized to increase the expression of the correctly folded anti-VEGFsc-dsFv on phage surface.

In order to further identify binding variants, more than 3000 colonieswere randomly selected from each of the libraries L2, L3, and L4 afterselection/amplification cycles for enrichment of the binding variants.These phage colonies were individually rescued and spotted onnitrocellulose membranes coated with VEGF (100 μg/30 ml). According tothe invention, each of the signal peptides having the amino acidsequences of SEQ ID NOS: 5-593 as listed in Tables 2, 3 and 4 wasobtained and proved to be capable of facilitating the expression of thesc-dsFv on phage surface. After normalization based on the standardphage solution signals in each of the blocks, the phage-displayed scFvexpression efficiency for each of the samples was calculated with thefollowing equation:

${Ratio} = {\frac{{sample}({CV})}{{sample}\left( {C\; 0} \right)}/\frac{{control}({CV})}{{control}\left( {C\; 0} \right)}}$

The value of the sample (CV) is the average normalized signal fromVEGF-coated membrane; that of the sample (C0) is the averaged normalizedsignal from the un-coated and un-blocked membrane. Similarly, those ofthe control (CV) and control (C0) are the averaged normalized signalsfor the control phage in the same block where the sample signals aremeasured on corresponding membrane. The ratio derived from the equationwas used to rank the efficiency of the sample phage binding to theimmobilized VEGF. All the phage samples with measurable bindingstrengths with the immobilized VEGF were ranked; the signal sequences ofthe top fifty ranked phage samples are shown and marked with “*” inTables 2-4.

Accordingly, new signal peptides that facilitate production ofdisulfide-stabilized single chain antibody were obtained (see Example2). In the embodiment of the invention, the signal peptide selected fromthe group consisting of the peptides having the amino acid sequences setforth in SEQ ID NOS: 5-593 were proved to facilitate production ofdisulfide-stabilized single chain antibody. On the other hand, a newisolated nucleic acid encoding the above mentioned signal peptide wasprovided as well.

In a preferred embodiment of the invention, the signal peptide selectedfrom the peptides having the amino acid sequences set forth in SEQ IDNOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304,381-429 and 476 was obtained and proved to facilitate production ofdisulfide-stabilized single chain antibody. Accordingly, the preferredisolated nucleic acid encoding each signal peptide as mentioned was alsoprovided.

In one example of the present invention, the anti-VEGF sc-dsFv wasdeveloped by using the signal peptides as identified and obtained by themethod of the present invention. In another example, anti-H5 sc-dsFvagainst influenza virus was developed (see FIG. 5).

In order to confirm the formation of disulfide bond in thephage-displayed sc-dsFv variants of the present invention, a fXasubstrate sequence (-IEGR-) in the linker sequence between the twovariable domains was constructed. As shown in FIG. 4, without the fXatreatment, both anti-VEGF scFv(fXa+) and scFv(fXa−) bound to immobilizedVEGF. In contrast, with the fXa treatment, only the anti-VEGF scFv(fXa−)bound to immobilized VEGF. The cleavage of the fXa substrate sequence inthe phage-displayed anti-VEGF scFv(fXa+) resulted in separation of thevariable domains, which in turn abolished the affinity of thephage-displayed scFv against immobilized VEGF. The anti-VEGF scFv(fXa−)was quite insensitive to the treatment of fXa, indicating that no otherfXa substrate sequences exist in the displayed protein.

Unexpectedly, it was found in the present invention that each of thesignal peptides having the amino acid sequences of SEQ ID NOS: 5-593 aslisted in Tables 2-4 enabled the expression and proper folding of thesc-dsFv structure on the phage-displayed platform. In addition, theyresulted in secretion of the soluble non-fusion sc-dsFv in culturemedia.

Accordingly, the present invention also provides a method for producinga disulfide-stabilized single chain antibody. The method includesproviding a host cell containing an expression construct, and culturingthe host cell in a medium under conditions allowing expression of thedisulfide-stabilized single chain antibody. The expression constructincludes a first nucleotide sequence encoding a signal peptide, and asecond nucleotide sequence encoding a single chain antibody capable offorming an interface disulfide bond. The second nucleotide sequence islocated 3′ downstream to the first nucleotide. The signal peptide hasthe amino acid sequence of:

(a) VKKLLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀AAQPAMAHHHHHHGH (SEQ ID NO:596), in whichX₁ is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X₂ is A, D, F, G, H, I,L, M, N, P, S, T, V, or W; X₃ is A, F, G, L, M, P, Q, R, S, T, V, or W;X₄ is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₅ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₆ is A, C, D, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, C, D, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, or Y; X₈ is A, C, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, C, D, E, F, G, H, I, L, M, N, P, Q,R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y;(b) VKKLLFAIPLX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀MAHHHHHHGH (SEQ ID NO:597), in whichX₁ A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X₂ is A, C, D, F,G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X₃ is A, C, D, F, G, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, or Y; X₅ is A, C, D, F, H, I, L, M, N, P, Q, R, S, T,V, W, or Y; X₆ is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, orY; X₇ is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A,C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X₉ is A, C, D, F, H, I, L,M, N, P, Q, R, S, T, V, W, or Y; and X₁₀ is A, C, D, E, F, G, H, L, M,P, Q, R, S, or T; or(c) VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH (SEQ ID NO:598), in whichX₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X₂ is A, C, D,F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X₃ is A, C, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ is A, C, D, F, G, H, I, L, M,N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X₁₀is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

Similar to the aforementioned experiment, the extent (percentage) of theinterface disulfide bond formation of the sc-dsFv from the optimumsignal sequence variants from L4 were tested. As shown in FIG. 6A,signal sequence optimization could improve the disulfide bond formationin the sc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule. Asshown in FIG. 6B, the interface disulfide bond formation enhanced theaffinity for the sc-dsFv-VEGF interaction.

In the present invention, a stability test of soluble sc-dsFv wasconducted. As shown in FIGS. 7A and 7B, the sc-dsFv antibody fragmentscaffold was indeed substantially more stable than the scFv scaffold dueto the interface disulfide bond in the sc-dsFv constructs.

According to the invention, the concentration of sc-dsFv antibodyproduced by the method disclosed herein was unexpectedly high, andstable. Thus, the present invention provides the sc-dsFv at a highconcentration sufficient for coating on a solid phase to produce anarray for detection or diagnosis without aggregation, different from theprior art where sc-Fv tends to precipitate under the same concentrationdue to aggregation.

Accordingly, the present invention provides an array ofdisulfide-stabilized single chain antibodies produced by theaforementioned method coated on a solid phase. In one example of theinvention, the solid phase may be made from silicon, plastic, nylon,glass, ceramic, photoresist or rubber. In one embodiment of the presentinvention, a microarray test was established using thedisulfide-stabilized single chain antibodies produced by the method ofthe invention, demonstrating that influenza virus could successfully bedetected by an array of a serious dilution of anti-H5 sc-dsFv coated ona glass.

The present invention is further illustrated by the following examples,which are provided for the purpose of demonstration rather thanlimitation.

EXAMPLES Preparation 1: VEGF Expression and Purification—Human VEGF-121

Human VEGF-121 (VEGF-A residue 34-135 receptor binding domain) (Fuh, G.et al., (2006). J. Biol. Chem., 281, 6625-6631) was expressed in E. colias inclusion body. The refolding and purification of VEGF-A were carriedout as described in Chang, H. J., et al., (2009) Structure, 17, 620-631.

Preparation 2: Phage Display Libraries with Diversified Signal SequencesN-Terminal to the sc-dsFv-pIII Fusion Protein

Phage display libraries with diversified sequences in the signal peptideregion N-terminal to the sc-dsFv-pIII fusion protein were constructedwith pCANTAB5E phagemid (GE-Amersham Biosciences) as shown in FIG. 1.Primers encoded with the sequence diversification shown in FIG. 1 weresynthesized by IDT (Integrated DNA Technologies).

For each of the phage display libraries, phagemid templates wereconstructed with TAA stop codons inserted in the sequence region fordiversification (Huang et al., (2010) J. Biol. Chem., in press). TheM13pIII-pelB signal sequence for phage-displayed pIII-fusion protein isa combination of the wild-type M13 signal peptide N-terminal to gene III(MKKLLFAIPLVVPFYSHS) (SEQ ID NO:594) and the pelB signal sequence ofPectobacterium wasabiae (MKYLLPTAAAGLLLLAAQPAMA) (SEQ ID NO:595). Thismerged signal sequence (shown in bold font above) was consideredcontaining the tentative n- h- and c-regions of the signal sequence. DNAlibraries were constructed to diversify the amino acid sequence in thekey regions. Each of the four of DNA libraries (L2, L3, L7) containedten consecutive NNK (N stands for 25% of G, C, A, and T, and K standsfor 50% of G and T; underlined by dashed lines) degenerate codonscovering a portion of the tentative signal sequence. Also shown in theFigure are the sequences containing TAA stop codons (underlined regions)used as the templates for the library constructions. Theoligonucleotide-directed mutagenesis procedure initially proposed byKunkel (Kunkel et al., (1987) Methods Enzymol, 154, 367-382) was usedfor the phagemid library construction. The TAA stop codons in thephagemid templates ensure that the un-mutated phagemid templates afterthe mutagenesis procedure are incapable of producing pIII fusion proteinfor phage surface display (Sidhu and Weiss, (2004) Construction phagedisplay libraries by oligonucleotide-directed mutagenesis. In: Clackson,T., and Lowman, H. B. (eds). Phage Display, 1st Ed., Oxford UniversityPress, New York).

After the oligonucleotide-directed mutagenesis procedure, E. coli strandER2738 was transformed with the phagemid libraries and the recombinantphage particles were rescued with helper phage M13KO7 (GE-AmershamBiosciences). The phage particles were precipitated with PEG/NaCl, andresuspended in PBS. More details of the phage library preparation can befound in a previous publication (Hsu, H. J. et al., (2008) J Biol Chem283(18), 12343-12353).

Seven sc-dsFv variants were constructed on the basis of the phagemidencoding the template anti-VEGF scFv(fXa+): S1(L:Gln38Cys & H:Gln39Cys);S2(L:Gly41Cys & H:Gly42Cys); S3(L:Ala43Cys & H:Gln112Cys); S4(L:Phe98Cys& H:Leu45Cys); S5(L:Gln100Cys & H:Gly44Cys); S6(L:Gln38Cys &H:Leu45Cys); S7(L:Ala43Cys & H:Gln112Cys & L:Gln100Cys & H:Gly44Cys).These cysteine pairs were determined by distance constrain for possibledisulfide bonds in the model structure (PDB code: 2FJG).

Preparation 3: Biopanning Against VEGF with Phage-Displayed Anti-VEGFsc-dsFv Libraries

Maxisorb Immune Tubes (Nunc) were coated with VEGF (25 μg in 1 ml PBS ineach tube) at 4° C. overnight. The tubes were blocked with 4 ml of 5%skim milk in PBST (PBS with 0.05% Tween 20) for one hour at roomtemperature with gentle shaking and then washed with PBST. In each ofthe tubes, 10¹¹ colony-forming units (cfu) of phage from each of thephage display libraries were mixed with 1 ml of 5% skim milk. The phageparticles were allowed to bind to the immobilized VEGF in the tube atroom temperature for two hours under gentle shaking. After the binding,the tubes were washed 10 times with PBST and 2 times with PBS. Onemilliliter of E. coli strand ER2738 in the log phase was added to eachof the tubes at room temperature with gentle shaking for 15 minutes.From each tube, the infected E. coli was transferred to 10 ml of a 2YTmedium containing 20 μg/ml of ampicillin and was titered with 2YT agarplates containing 100 μg/ml of ampicillin. The infected E. coli wasincubated at 37° C. for one hour with vigorous shaking Ampicillin wasthen added to reach final concentration of 100 μg/ml. The culture wasincubated for another hour at 37° C. before transferred to final 100 ml2YT medium (100 μg/ml of ampicillin) containing 10¹¹ cfu M13KO7 helperphage. After two hours of incubation, kanamycin was added to finalconcentration of 70 μg/ml. The culture was incubated at 37° C. overnightwith vigorous shaking. The phage in the supernatant of the culture washarvested by centrifugation. The phage was titered, precipitated withPEG/NaCl, and resuspended in PBS. The phage solution was ready for thenext round of selection.

Preparation 4: Enzyme-Linked Immunosorbant Assay (ELISA) forPhage-Displayed Anti-VEGF sc-dsFv Binding Against Immobilized VEGF andAnti-E-Tag Antibody

Single E. coli colonies harboring the selected phagemids were randomlypicked using a GENETIX Qpix II colony picker to 96-well deep wellculture plates. Each well contained 960 μl 2YT (100 μg/ml of ampicillinand 10 μg/ml of tetracyclin). The culture plates were incubated at 37°C. shaking vigorously for 4 hours before adding 20 μl of M13KO7 helperphage (10¹¹ cfu/ml). The plates were then incubated at 37° C. for onehour with vigorous shaking before adding 20 μl of kanamycin to the finalconcentration of 50 μg/ml. After overnight incubation at 37° C. withvigorous shaking, the cultures were centrifuged at 3000 g for 10 minutesat 4° C. From each well of the culture plates, 100 μl of the supernatantwas mixed with 100 μl of 5% skim milk. Half of the phage mixture wasadded to a corresponding well of a 96-well Maxisorb microtiter plateprecoated with VEGF (1 μg/well) and blocked with 5% skim milk; the otherhalf was added to a corresponding well of another microtiter plateprecoated with polyclonal goat anti-E-tag antibody (1 μg/well, NovusBiologicals). After one hour incubation at room temperature, the ELISAplates were washed six times with PBST. The phage particles remained onthe plates were measured with HRP-labeled mouse anti-M13 antibody(1/3000, GE Healthcare) and TMB substrate (KPL). The reaction wasstopped with 50 μl of 1 N HCl and the signal intensity was measured atOD 450 nm.

Preparation 5: Measurement of Interface Disulfide Bond Formation inPhage-Displayed Anti-VEGF sc-dsFv

Fifty microliters of a freshly prepared phage supernatant (see above)was mixed with 50 μl of a two-fold concentrated reaction buffercontaining 1 unit of bovine factor Xa (fXa) (Novagen) in a Maxisorbmicrotiter plate precoated with VEGF (1 μg/well) and blocked with 5%skim milk. After two hours of enzymatic reaction at 37° C., the phageparticles remained bound to the microtiter plate were measured followingthe same ELISA procedure as described above.

Preparation 6: Western Blot Assay for the Phage-Displayed Anti-VEGFsc-dsFv

Single colony phage was amplified, harvested, precipitated withPEG/NaCl, and resuspended in PBS (see above). Phage particles (10¹¹ cfu)were prepared under either a non-reducing or reducing condition beforeelectrophoresis in a 10% SDS-polyacrylamide gel. After theelectrophoresis, the proteins in the gel were transferred onto apolyvinylidene fluoride (PVDF) membrane (Millipore). The membrane wasblocked with 5% skim milk for 1 hour at room temperature and thenincubated with a monoclonal mouse anti-pIII antibody (1/3000 mg/ml, NewEngland Biolabs) for one hour at room temperature. After three washes (5minutes each) with PBST, the membrane was incubated with HRP-labeledanti-mouse antibody (1/3000, GE Healthcare) for 1 hour at roomtemperature. After three washes with 10 ml PBST, the membrane wasdeveloped with 4-chloro-1-naphthol (4CN) substrate (KPL) until thedesired color intensity was achieved.

Preparation 7: Preparation of Non-Fusion Soluble scFv/sc-dsFv

Seven hundred and fifty microliters of mid-log phase (OD_(600 nm)=0.6)E. coli host (non-suppressor strain HB2151 or suppressor strain ER2738)grown in a 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/LNaCl, pH 7.0) was infected with 50 μl of a phage solution (10¹¹ cfu/ml).After one hour incubation at 37° C. with shaking, 100 μl ampicillin in a2YT medium was added to the final concentration of 100 μg/ml. Afteranother hour of incubation, 100 μlisopropyl-beta-D-thiogalactopyranoside (IPTG) in a 2YT medium was addedto the final concentration of 1 mM. The culture was kept at 37° C. withvigorous shaking overnight. The secreted soluble scFv or sc-dsFv in thesupernatant was separated from the bacterial host by centrifugation at3000 g for 10 minutes.

Preparation 8: ELISA for Immobilized VEGF Binding

For phage ELISA, each well in a Maxisorb 96-well microtiter plate (Nunc)was coated with 2 μg VEGF at 4° C. overnight. The wells were blockedwith 5% skim milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mMKH₂PO₄, 0.1% tween20, pH 7.4) for one hour. After 3×300 μl PBST and2×300 μl PBS washes, 100 μl of a phage solution and 100 μl of 5% skimmilk in PBST were added to each well and incubated at room temperaturewith shaking for one hour. After washing each of the wells three timeswith 300 μl of PBST each and twice with 300 μl of PBS each, the boundphages were labeled with anti-M13 antibody conjugated with HRP(GE-Amersham) 1/3000 dilution in 5% skim milk in PBST for one hour. TheELISA signal was developed by incubating each well with 100 μl of a TMBsolution (KPL Inc.) for 5 minutes. The reaction was stopped with 100 μlN HCl, and the optical density was recorded with VICTOR3 MultilabelPlate Readers (Perkin Elmer) at 450 nm.

For scFv or sc-dsFv ELISA, 100 μl of a soluble scFv solution was usedinstead of phage solution, and HRP-conjugated protein L (0.5 μg/ml in 5%skim milk in PBS, from Pierce) was used instead of HRP-conjugatedanti-M13 antibody. When needed, the ELISA signals were normalized withthe signals of the control anti-VEGF scFv in serial dilution.

Preparation 9: fXa Protease Digestion

For phage solutions, 20 μl (1 unit) of bovine factor Xa (fXa) protease(Novagen) in a six-fold concentrated reaction buffer was added to 100 μlof a phage solution at 37° C. After 2 hours of enzymatic reaction, 100μl of 5% skim milk in PBST was added to the reaction mixture before theVEGF-binding ELISA measurement was carried out in the manner describedin the previous section. The fXa resistance percentage was calculatedwith the ratio of the ELISA reading in the presence of fXa over theELISA reading in the absence of fXa. The ELISA readings for the ratiowere adjusted by shift the baseline determined with the null controlELISA readings. For soluble scFv/sc-dsFv fXa digestion, all procedureswere the same except that the enzymatic reaction was carried out for onehour at room temperature.

Preparation 10: Construction of Anti-H5 sc-dsFv Against Influenza Virus

The construction of scFv library derived from mouse spleen afterimmunization of hemagglutinin from influenza virus was based on theprotocols described in “Phage Display, A Laboratory Manual, edited byCarlos F. Barbas Ill, Dennis R. Burton, Jamie K. Scott, and Gregg J.Silverman, 2001, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA”. In brief, after hemagglutinin immunization, thetotal RNA derived from mouse spleen was purified by Trizol reagent(Invitrogen) according to the manufacturer's protocols. After cDNAsynthesis by reverse transcriptase, the gene fragments encoded heavy andlight chains of antibody variable region were amplified by the specificprimer sets described in the book mentioned above, respectively. ThescFv fragments were synthesized by two-steps PCR reactions, and thencloned into a pCANTAB 5E phagemid vector with the signal sequencederived from the library 2 for sc-dsFv phage production. The librarycomplexity was 4.5×10⁷. After panning against H5, two clones wereselected for mono-spectral binding to H5 (clone 8a) and broad spectralbinding to H1, H3 and H5 (clone 12a). These two clones were subjected todisulfide bond formation mutants between L100 and H44 (based on Kabatnumbering) and then for sc-dsFv phage production and ELISA detection(8aS5 and 12aS5, respectively).

Preparation 11: Microarray Test for sc-dsFv Binding of H5 InfluenzaVirus

The just-described sc-dsFv ds-8a or and ds-12a was subcloned into apET32a vector with thioredoxin as a fusion protein partner at theN-terminus. The thioredoxin-sc-dsFv fusion proteins could be expressedin Rosetta-gami B strain of E. coli in a soluble form. Afterpurification and TEV protease digestion to remove thioredoxin, thepurified ds-8a protein was found to have a binding affinity andspecificity similar to those of H5 based on an ELISA assay and arraystudies. The protein ds-AV1, ds-12a and ds-8a were spot on glass slidescoated with streptavidin as the purified ds-AV1, ds-12a and ds-8aproteins contained a biotinylated Avitag sequence at their C-termini.The highest protein concentration used for this protein array was 8mg/ml, 8 mg/ml, and 0.8 mg/ml, respectively. These proteins were 2 folddilution with a 100 mM sodium phosphate buffer, pH 8.5 from the highestprotein concentration for 15 times for spotting (10 nl/spot), and thenthe protein of each concentration was spotted for 5 replicates. Afterspotting, each glass slide was sealed to form 16 distinct squares forreaction. After blocking with 5% BSA for 30 minutes, the H5 influenzavirus (about ˜10⁷ PFU/ml) was added to react with spotted sc-dsFvs for30 minutes. After 3 times wash with a phosphate buffer for 5 minuteseach, 40 nm fluorescence beads coated with ds-8a (˜10⁷) were added toeach square and incubated for 30 minutes. After 3 times wash again witha phosphate buffer for 5 minutes each, the glass array was air-dried fordetection.

Example 1 Selection of Signal Sequences for Effective Expression ofAnti-VEGF sc-dsFv on M13 Phage Surface

Phage display libraries L2, L3, and L4 were constructed to diversify thesignal sequence of the S5 anti-VEGF sc-dsFv-pIII fusion protein as shownin FIG. 1. The complete DNA construct and the amino acid sequence of theS5 anti-VEGF sc-dsFv are shown in FIG. 1. The S5 sequence remainedunchanged in all the variants from the libraries. The complexities ofthe L2, L3, and L4 phage display libraries were 3.1×10⁹, 3.7×10⁹, and1.5×10⁹, respectively. These libraries were designed to diversify thesignal peptide sequences in the h-region, c-region, and a few N-terminalresidues of the mature phage-displayed anti-VEGF sc-dsFv.

Each of the phage display libraries was selected for binding againstimmobilized VEGF. The trends of enrichment of the VEGF-binding phagevariants from each of the three libraries, plotted as functions of thenumber of selection/amplification cycle, are shown in FIG. 2. After fourrounds of selection/amplification cycle, the VEGF-binding phage variantswere enriched for more than one order of magnitude. The enrichmenttrends were similar among the variants from the three libraries. Thisresult indicates that the signal sequence regions covered by the threesignal sequence libraries (FIG. 1) could all be optimized to increasethe expression of the correctly folded anti-VEGF sc-dsFv on phagesurface.

Example 2 Interface Disulfide Bond Formation in Anti-VEGF sc-dsFv on M13Phage Surface

In order to test the formation of the disulfide bond in thephage-displayed sc-dsFv variants, we constructed two controlphage-displayed anti-VEGF scFv variants: one with a factor Xa cuttingsite, -IEGR- (SEQ ID NO:599), encoded in the linker peptide connectingthe two variable domains (anti-VEGF scFv(fXa+)); the other without thisfXa cutting site (anti-VEGF scFv(fXa−)). As shown in FIG. 3, the S5anti-VEGF sc-dsFv was constructed with a fXa substrate sequence (-IEGR-)in the linker sequence between the two variable domains. The cleavage ofthe fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+)resulted in separation of the variable domains, which in turn abolishedthe affinity of the phage-displayed scFv against immobilized VEGF. Bothphage-displayed scFv's did not have the engineered interface disulfidebond as in S5; the scFv(fXa+) construct had the -IEGR- (SEQ ID NO:599)site in the linker peptide (-(G)₄SIEGRS(G)₄S-) (SEQ ID NO:600), whilethe scFv(fXa−) construct had the conventional -(G)₄S(G)₄S(G)₄S- (SEQ IDNO:601) linker peptide.

As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+)and scFv(fXa−) bound to immobilized VEGF. But with the fXa treatment,only the anti-VEGF scFv(fXa−) bound to immobilized VEGF. In contrast,all the S5 signal sequence variants for the phage-displayed sc-dsFvshowed substantial increase in resistance to fXa protease activity,indicating that the interface disulfide bonds in the anti-VEGF sc-dsFv'swere formed to stabilize the functional dimeric structure after thecleavage of the peptide linker between the two variable domains. Theresults unambiguously demonstrated that the engineered interfacedisulfide bond was correctly formed in the phage-displayed S5 anti-VEGFsc-dsFv from some of the signal sequence variants from all threeVEGF-binding enriched signal sequence libraries (L2, L3 and L4).

Example 3 Preference Sequence Patterns of the Optimum Signal Peptides inEffective Expression of Functional Anti-VEGF sc-dsFv

The functionality of the anti-VEGF sc-dsFv on phage surface wasquantified with two quantitative measurements: the affinity of thesc-dsFv against VEGF and the extent of the interface disulfide bondformation. After the tests, 250, 126, and 213 optimum signal sequenceswere found in L2, L3, and L4 library, respectively, which are summarizedas following Tables 2-4. Among them, fifty signal sequence variants withthe highest sc-dsFv-VEGF binding affinities selected from more than 3000random single colonies of the enriched libraries L2, L3, and L4 weremarked with the symbol “*.” The symbol “q” indicated that the nucleotidesequence TAG (amber stop codon) that could be translated to Gln (Q) with0.8˜20% in E. coli amber suppressor strains which were normally used inphage production.

TABLE 2 Preference sequence patterns se1ected from L2 S5 sc-dsFv 1ibraryNo.  Code Sequence SEQ ID NO: M13-pe1B VKKLL FAIPLVVPFY AAQPAMAHHHHHH 4  1 1.12B VKKLL VLSHLPFMTD AAQPAMAHHHHHH * 5   2 9.26.10B VKKLLSHWLLSSqLQ AAQPAMAHHHHHH * 6     3 2.12A VKKLL AMSLAPSVFPAAQPAMAHHHHHH * 7   4 9.12A VKKLL WSLFFqqLNP AAQPAMAHHHHHH * 8   5 2.12FVKKLL LLSLLQRPLP AAQPAMAHHHHHH * 9   6 1.2H VKKLL LSSWLMTRFPAAQPAMAHHHHHH * 10   7 6.9G VKKLL VLSHFPAFVP AAQPAMAHHHHHH * 11   8 1.8FVKKLL PLLSLPLPPN AAQPAMAHHHHHH * 12   9 7.1B VKKLL VLTPMHFSSPAAQPAMAHHHHHH * 13  10 9.26.10A VKKLL ILALPQSYPL AAQPAMAHHHHHH * 14  115.4A VKKLL qALYFSLPSS AAQPAMAHHHHHH * 15  12 YJ2.2 VKKLL VSAMTSASFPAAQPAMAHHHHHH * 16  13 5.2F VKKLL LPASWLFGQP AAQPAMAHHHHHH 17  14 10.2DVKKLL WSLFFqqLNP AAQPAMAHHHHHH * 18  15 YJ2.34 VKKLL VVMALRSSAPAAQPAMAHHHHHH * 19  16 3.3F VKKLL FLWPFYNGHI AAQPAMAHHHHHH 20  17 4.1AVKKLL QSFYLSLqLD AAQPAMAHHHHHH * 21  18 10.7H VKKLL SLTFPFTIHSAAQPAMAHHHHHH * 22  19 1.9D VKKLL WPVLSPSLFP AAQPAMAHHHHHH * 23  205.12D VKKLL PWLFSTFPSS AAQPAMAHHHHHH * 24  21 1.8D VKKLL IMSSLPTLSPAAQPAMAHHHHHH * 25  22 4.11F VKKLL IMSRVLAPDF AAQPAMAHHHHHH * 26  231.7C VKKLL FDFWFSSFLq AAQPAMAHHHHHH * 27  24 4.8G VKKLL YGqLMLLSSDAAQPAMAHHHHHH * 28  25 4.4E VKKLL PWLFPFHAYP AAQPAMAHHHHHH * 29  261.12G VKKLL LVMTLSRQPF AAQPAMAHHHHHH 30  27 4.8A VKKLL ASAYLYHGLSAAQPAMAHHHHHH * 31  28 4.4C VKKLL PFFAGVLqHP AAQPAMAHHHHHH * 32  293.11A VKKLL ALSSPFFHIP AAQPAMAHHHHHH * 33  30 10.3F VKKLL PTRqPMMYPPAAQPAMAHHHHHH * 34  31 YJ2.15 VKKLL QLLMPFLNSP AAQPAMAHHHHHH * 35  329.9H VKKLL CSLGYACIPP AAQPAMAHHHHHH * 36  33 4.9C VKKLL LMPWLFNSPPAAQPAMAHHHHHH 37  34 3.12B VKKLL LDqLAYAALS AAQPAMAHHHHHH * 38  35 4.10GVKKLL qSTVFFSWLS AAQPAMAHHHHHH * 39  36 YJ2.18 VKKLL LPWALSHQVLAAQPAMAHHHHHH * 40  37 7.2E-q VKKLL ALTYPAFLYD AAQPAMAHHHHHH * 41  381.11A VKKLL AMAPPMMSMN AAQPAMAHHHHHH * 42  39 5.3D VKKLL WWSSLFAPSPAAQPAMAHHHHHH 43  40 4.6H VKKLL GSFILARSMD AAQPAMAHHHHHH 44  41 5.11CVKKLL MVLTSWHPYP AAQPAMAHHHHHH * 45  42 2.8C VKKLL FSLRFFFPSSAAQPAMAHHHHHH 46  43 2.5F VKKLL WLWSTPLFPH AAQPAMAHHHHHH 47  44 2.2AVKKLL PLLFSLDGDP AAQPAMAHHHHHH * 48  45 3.2C-d VKKLL SVSLSSYSFYAAQPAMAHHHHHH * 49  46 3.1H VKKLL LNGTESAqLF AAQPAMAHHHHHH * 50  47 6.4AVKKLL WHVLPYLPNS AAQPAMAHHHHHH * 51  48 4.10E VKKLL SIVPLFSPqSAAQPAMAHHHHHH * 52  49 7.4H VKKLL VMTSPMLAPG AAQPAMAHHHHHH * 53  50 2.5HVKKLL VLSLPSIAPH AAQPAMAHHHHHH 54  51 6.4E VKKLL qSLLLLRALLAAQPAMAHHHHHH * 55  52 2.1A VKKLL FSLPVFFDLP AAQPAMAHHHHHH 56  53 4.11DVKKLL LLFSMARPLP AAQPAMAHHHHHH * 57  54 7.10A VKKLL TqAVFPFTFNAAQPAMAHHHHHH * 58  55 3.2E VKKLL LASWLFRADM AAQPAMAHHHHHH * 59  56 5.2EVKKLL PFLFPFPSPS AAQPAMAHHHHHH * 60  57 YJ2.128 VKKLL ALSAWSLSQTAAQPAMAHHHHHH * 61  58 4.7H VKKLL ALLPLFPTqH AAQPAMAHHHHHH * 62  592.10F VKKLL AALASFPPAP AAQPAMAHHHHHH * 63  60 YJ2.22 VKKLL LLMPFLNQSPAAQPAMAHHHHHH * 64  61 7.5A VKKLL FTSGLKLVPP AAQPAMAHHHHHH 65  62 6.10FVKKLL LqPLLSIYLN AAQPAMAHHHHHH 66  63 4.11B VKKLL LSSLWSAYMDAAQPAMAHHHHHH 67  64 2.5C VKKLL LLGqSLMHFQ AAQPAMAHHHHHH 68  65 YJ2.25VKKLL PQLAMSLPSI AAQPAMAHHHHHH 69  66 10.3H VKKLL YETMLSSYLYAAQPAMAHHHHHH 70  67 3.10D VKKLL SLYYFPLVPY AAQPAMAHHHHHH 71  68 4.7CVKKLL qRTVAAAYFW AAQPAMAHHHHHH 72  69 4.12D VKKLL FLTWLRYGFPAAQPAMAHHHHHH 73  70 6.1A VKKLL LLLTLMqPTS AAQPAMAHHHHHH 74  71 8.10CVKKLL FDFFTHVHLF AAQPAMAHHHHHH 75  72 5.6E VKKLL ALYPHFVSFTAAQPAMAHHHHHH 76  73 4.11E VKKLL LPYAIqLFSP AAQPAMAHHHHHH 77  74 YJ2.5VKKLL WFPLHSSLLP AAQPAMAHHHHHH 78  75 4.7A VKKLL PALLLATAAFAAQPAMAHHHHHH 79  76 3.11C VKKLL LASVAWNLDS AAQPAMAHHHHHH 80  77 YJ2.121VKKLL VGSLLFWPQQ AAQPAMAHHHHHH 81  78 4.5F VKKLL SPLLFLqNYTAAQPAMAHHHHHH 82  79 3.2F VKKLL SYWLDFIqVL AAQPAMAHHHHHH 83  80 10.3CVKKLL VPSFLLSPSP AAQPAMAHHHHHH 84  81 9.23.7H VKKLL SLYWLTSqPLAAQPAMAHHHHHH 85  82 3.9A VKKLL FALSSVHSPP AAQPAMAHHHHHH 86  83 4.11HVKKLL SYYSLLYSYP AAQPAMAHHHHHH 87  84 3.1C VKKLL LVSGLqPWYFAAQPAMAHHHHHH 88  85 2.5A VKKLL VLATPLHLSP AAQPAMAHHHHHH 89  86 10.6H-qVKKLL SLAFPLFTPP AAQPAMAHHHHHH 90  87 3.6A VKKLL SLVPIFPFSTAAQPAMAHHHHHH 91  88 8.10D VKKLL qPVLFSFFIR AAQPAMAHHHHHH 92  89 4.3BVKKLL MSqFLNLLSP AAQPAMAHHHHHH 93  90 2.3G VKKLL WAVqPLFPLNAAQPAMAHHHHHH 94  91 5.3H VKKLL MFSLVPSPPI AAQPAMAHHHHHH 95  92 10.7BVKKLL PFFLQPFqFP AAQPAMAHHHHHH 96  93 7.2D-q VKKLL PDLLASVLPVAAQPAMAHHHHHH 97  94 2.9H VKKLL FWqFLWPSLP AAQPAMAHHHHHH 98  95 6.4AVKKLL LLGqFFPNPM AAQPAMAHHHHHH 99  96 6.4D VKKLL TLSALSQWHPAAQPAMAHHHHHH 100  97 9.4D VKKLL SLVYFFPFYP AAQPAMAHHHHHH 101  98 10.2HVKKLL FAFAPAPFYH AAQPAMAHHHHHH 102  99 4.12B VKKLL FLPFALVPRQAAQPAMAHHHHHH 103 100 4.1F VKKLL ALWMqLYPQD AAQPAMAHHHHHH 104 101 YJ2.27VKKLL ASILFSHAAP AAQPAMAHHHHHH 105 102 2.2C VKKLL LPLPWSLHLYAAQPAMAHHHHHH 106 103 4.9C VKKLL LPHFMSFWFE AAQPAMAHHHHHH 107 104 7.3EVKKLL LFQPFWPIPY AAQPAMAHHHHHH 108 105 4.7F VKKLL LLFSLGRLPPAAQPAMAHHHHHH 109 106 7.12G VKKLL PLWVLLKDPL AAQPAMAHHHHHH 110 107 9.3BVKKLL MSFATLFPHN AAQPAMAHHHHHH 111 108 4.5B VKKLL qHSLVTSWLCAAQPAMAHHHHHH 112 109 5.2H VKKLL LLFqGAFVGq AAQPAMAHHHHHH 113 110 4.4CVKKLL WMFHSLPFSP AAQPAMAHHHHHH 114 111 6.8G VKKLL LTqLLLTRLHAAQPAMAHHHHHH 115 112 4.10A VKKLL ALTLVPSSYP AAQPAMAHHHHHH 116 113 4.5DVKKLL LPWYMLLSDS AAQPAMAHHHHHH 117 114 9.3E VKKLL VVTqFWPSLPAAQPAMAHHHHHH 118 115 4.3G VKKLL LSTLFLWHVR AAQPAMAHHHHHH 119 116 9.7EVKKLL RSLFFqqLYP AAQPAMAHHHHHH 120 117 YJ2.30 VKKLL TLTTLHQTFPAAQPAMAHHHHHH 121 118 1.3B VKKLL SALLAPWYWD AAQPAMAHHHHHH 122 119 8.9BVKKLL AIqqRMQIYT AAQPAMAHHHHHH 123 120 3.4E VKKLL LLFPWFQPPYAAQPAMAHHHHHH 124 121 9.23.7E VKKLL YFTSLLGqFP AAQPAMAHHHHHH 125 1226.3D VKKLL PVLIFLSEIR AAQPAMAHHHHHH 126 123 9.5G VKKLL VATSLRWAVTAAQPAMAHHHHHH 127 124 YJ2.54 VKKLL AQLFHLFATH AAQPAMAHHHHHH 128 125 8.6GVKKLL LqFSALFNSF AAQPAMAHHHHHH 129 126 7.12C-q VKKLL FHLMSMLPPPAAQPAMAHHHHHH 130 127 5.4C VKKLL PVCSqSMFPI AAQPAMAHHHHHH 131 128 YJ2.48VKKLL LLLSSSYQSP AAQPAMAHHHHHH 132 129 4.3D VKKLL LDSLFFHAPLAAQPAMAHHHHHH 133 130 7.7A VKKLL qAWVFSAHQL AAQPAMAHHHHHH 134 131 YJ2.99VKKLL FQALGALTSP AAQPAMAHHHHHH 135 132 9.9D VKKLL CFFFFLqFHPAAQPAMAHHHHHH 136 133 4.12F-f VKKLL CFSHLALPSP AAQPAMAHHHHHH 137 1346.2B VKKLL FGSWIPFTQM AAQPAMAHHHHHH 138 135 4.6F VKKLL GLGYFNWTLLAAQPAMAHHHHHH 139 136 10.4A VKKLL HLFPLFQFHH AAQPAMAHHHHHH 140 137 5.6BVKKLL SEHVSSICVL AAQPAMAHHHHHH 141 138 3.11E VKKLL FSCLLDPTCPAAQPAMAHHHHHH 142 139 8.3F VKKLL LYLLHPSFLP AAQPAMAHHHHHH 143 140 2.2FVKKLL WCAPLLYSLR AAQPAMAHHHHHH 144 141 2.3F VKKLL FAMFPYTFqTAAQPAMAHHHHHH 145 142 10.5D VKKLL LPSLFYVESL AAQPAMAHHHHHH 146 143 8.8BVKKLL SLWLSSLSVL AAQPAMAHHHHHH 147 144 YJ2.17 VKKLL PHLWFLWSLKAAQPAMAHHHHHH 148 145 7.5B VKKLL ASDPVWYFLW AAQPAMAHHHHHH 149 146 10.12DVKKLL GLPLMGLqSL AAQPAMAHHHHHH 150 147 2.4H VKKLL PQLLLLRALSAAQPAMAHHHHHH 151 148 5.5D VKKLL APSAFSLHLF AAQPAMAHHHHHH 152 149 9.4CVKKLL FqLSSLFVPY AAQPAMAHHHHHH 153 150 4.5H VKKLL VPSFLSTMIEAAQPAMAHHHHHH 154 151 2.7B VKKLL ASPFFASYLW AAQPAMAHHHHHH 155 152 YJ2.23VKKLL LQYLLSPIGY AAQPAMAHHHHHH 156 153 6.2D VKKLL VLSVPISAHHAAQPAMAHHHHHH 157 154 7.4A VKKLL MMqALSSLPE AAQPAMAHHHHHH 158 155 4.12BVKKLL MPAVLATRLT AAQPAMAHHHHHH 159 156 6.12E VKKLL PFTAWIIDGWAAQPAMAHHHHHH 160 157 YJ2.125 VKKLL TQLLPLWQPL AAQPAMAHHHHHH 161 158YJ2.21 VKKLL LVPSLLPLTQ AAQPAMAHHHHHH 162 159 10.12B VKKLL PIqSCMVIPSAAQPAMAHHHHHH 163 160 YJ2.35 VKKLL WSLHLATRLL AAQPAMAHHHHHH 164 1616.11H VKKLL qQVLLCSTLR AAQPAMAHHHHHH 165 162 7.3B VKKLL LLRYFLDPMYAAQPAMAHHHHHH 166 163 10.12A VKKLL IPQFLRSHHR AAQPAMAHHHHHH 167 164YJ2.6 VKKLL GVLHLALSLR AAQPAMAHHHHHH 168 165 4.12C VKKLL LVTSqFSLVPAAQPAMAHHHHHH 169 166 YJ2.19 VKKLL PLALSWFQLR AAQPAMAHHHHHH 170 167YJ2.88 VKKLL QHQWYPTVLM AAQPAMAHHHHHH 171 168 YJ2.29 VKKLL LMYWLSKPLSAAQPAMAHHHHHH 172 169 YJ2.8 VKKLL TQLTLSSSPI AAQPAMAHHHHHH 173 170YJ2.94 VKKLL QLTALLSRLI AAQPAMAHHHHHH 174 171 YJ2.107 VKKLL LMTFGTTPQSAAQPAMAHHHHHH 175 172 YJ2.133 VKKLL SAFSFSLSST AAQPAMAHHHHHH 176 1736.1A VKKLL APWLVLPHFP AAQPAMAHHHHHH 177 174 YJ2.81 VKKLL HVLSFAPPMPAAQPAMAHHHHHH 178 175 YJ2.38 VKKLL NWLFFAHPFS AAQPAMAHHHHHH 179 176YJ2.20 VKKLL QLAVLLGSLR AAQPAMAHHHHHH 180 177 7.1D VKKLL LFGLFYFRACAAQPAMAHHHHHH 181 178 YJ2.98 VKKLL FQFFVVWRLL AAQPAMAHHHHHH 182 179YJ2.39 VKKLL PWAWPPPPFW AAQPAMAHHHHHH 183 180 YJ2.130 VKKLL LQLVIVYYLRAAQPAMAHHHHHH 184 181 YJ2.16 VKKLL RQSVLLSALH AAQPAMAHHHHHH 185 1823.12E VKKLL VYGYFLTTFR AAQPAMAHHHHHH 186 183 YJ2.53 VKKLL CFSPLFGFHTAAQPAMAHHHHHH 187 184 YJ2.100 VKKLL PGYALWQTI PAAQPAMAHHHHHH 188 185YJ2.58 VKKLL QRIFICFFLR AAQPAMAHHHHHH 189 186 8.2A VKKLL PHVFSCqLSAAAQPAMAHHHHHH 190 187 5.10A VKKLL SPLSLSVKLL AAQPAMAHHHHHH 191 188 9.2DVKKLL ARSLFSGSML AAQPAMAHHHHHH 192 189 YJ2.92 VKKLL LQFLIVFPLRAAQPAMAHHHHHH 193 190 YJ2.32 VKKLL LAVLLGQSLR AAQPAMAHHHHHH 194 191YJ2.14 VKKLL LLSHLFLRLH AAQPAMAHHHHHH 195 192 8.4E VKKLL LAMVFFVTLRAAQPAMAHHHHHH 196 193 YJ2.117 VKKLL WLFALPQENV AAQPAMAHHHHHH 197 194YJ2.66 VKKLL HPLVLLSSSP AAQPAMAHHHHHH 198 195 YJ2.131 VKKLL LQYLFMLSMRAAQPAMAHHHHHH 199 196 4.11H VKKLL PALLIRYASV AAQPAMAHHHHHH 200 197YJ2.78 VKKLL QQFTSPFLLL AAQPAMAHHHHHH 201 198 YJ2.44 VKKLL SPCFFLLYLRAAQPAMAHHHHHH 202 199 YJ2.90 VKKLL PGMPLFFTNS AAQPAMAHHHHHH 203 200YJ2.47 VKKLL PQVFFLFRPF AAQPAMAHHHHHH 204 201 YJ2.110 VKKLL PFPILLQSPFAAQPAMAHHHHHH 205 202 YJ2.74 VKKLL FQACCLFPLQ AAQPAMAHHHHHH 206 203YJ2.55 VKKLL AVVHTMPLFS AAQPAMAHHHHHH 207 204 YJ2.108 VKKLL QFSWAFVSILAAQPAMAHHHHHH 208 205 YJ2.96 VKKLL PVCLFWSFFR AAQPAMAHHHHHH 209 206YJ2.70 VKKLL QLLWQQQVPV AAQPAMAHHHHHH 210 207 YJ2.60 VKKLL PLQALSWFLRAAQPAMAHHHHHH 211 208 YJ2.119 VKKLL FYLLCRLSLQ AAQPAMAHHHHHH 212 209YJ2.82 VKKLL YLQILVICLR AAQPAMAHHHHHH 213 210 YJ2.63 VKKLL QLFLIVFPLRAAQPAMAHHHHHH 214 211 10.5A VKKLL PLHFALFFRL AAQPAMAHHHHHH 215 212YJ2.85 VKKLL PFPMHLVLPF AAQPAMAHHHHHH 216 213 YJ2.86 VKKLL PLLFSPPSLHAAQPAMAHHHHHH 217 214 YJ2.126 VKKLL CQSITFSSIW AAQPAMAHHHHHH 218 215YJ2.112 VKKLL WQRLFPFLLI AAQPAMAHHHHHH 219 216 YJ2.77 VKKLL MVPFWPFSFTAAQPAMAHHHHHH 220 217 YJ2.103 VKKLL QAFPLPPLLV AAQPAMAHHHHHH 221 218YJ2.134 VKKLL PLYLLFRSFV AAQPAMAHHHHHH 222 219 YJ2.91 VKKLL HRSMYLSWLYAAQPAMAHHHHHH 223 220 YJ2.64 VKKLL LLSTLVRAPY AAQPAMAHHHHHH 224 221YJ2.87 VKKLL PLALSQWFLR AAQPAMAHHHHHH 225 222 YJ2.116 VKKLL AQGMIFFLRLAAQPAMAHHHHHH 226 223 YJ2.62 VKKLL FCCRLALQFF AAQPAMAHHHHHH 227 224YJ2.102 VKKLL YLQFLSLMLS AAQPAMAHHHHHH 228 225 YJ2.106 VKKLL CQATFPTLLCAAQPAMAHHHHHH 229 226 YJ2.124 VKKLL ARSYLYFSLS AAQPAMAHHHHHH 230 227YJ2.111 VKKLL YQSSFLPLFW AAQPAMAHHHHHH 231 228 YJ2.104 VKKLL SASFLAFRITAAQPAMAHHHHHH 232 229 YJ2.67 VKKLL SVLFLSHYHS AAQPAMAHHHHHH 233 230YJ2.105 VKKLL PLALLYVRLS AAQPAMAHHHHHH 234 231 YJ2.127 VKKLL PEFLLLFRFFAAQPAMAHHHHHH 235 232 YJ2.80 VKKLL FPSLYAWGGL AAQPAMAHHHHHH 236 233YJ2.122 VKKLL LQAAAFFCWL AAQPAMAHHHHHH 237 234 YJ2.79 VKKLL PFFLFCSSLRAAQPAMAHHHHHH 238 235 YJ2.115 VKKLL ELTQLWLFHL AAQPAMAHHHHHH 239 236YJ2.113 VKKLL PGVPLLLCFR AAQPAMAHHHHHH 240 237 YJ2.114 VKKLL SQAYLSYFLYAAQPAMAHHHHHH 241 238 YJ2.61 VKKLL ISYAFLVRVT AAQPAMAHHHHHH 242 239YJ2.123 VKKLL APALLRSILA AAQPAMAHHHHHH 243 240 YJ2.109 VKKLL HSHTLLMSLHAAQPAMAHHHHHH 244 241 YJ2.83 VKKLL AVSAFVSLVR AAQPAMAHHHHHH 245 242YJ2.31 VKKLL TLITFKFLPH AAQPAMAHHHHHH 246 243 YJ2.49 VKKLL QQFAIPLVEFAAQPAMAHHHHHH 247 244 YJ2.75 VKKLL MPCLLVYYLE AAQPAMAHHHHHH 248 245YJ2.71 VKKLL RYCLLLQIVR AAQPAMAHHHHHH 249 246 YJ2.45 VKKLL SLALLRVSLGAAQPAMAHHHHHH 250 247 YJ2.68 VKKLL IIGRIALILR AAQPAMAHHHHHH 251 248YJ2.24 VKKLL PQLICAFILR AAQPAMAHHHHHH 252 249 8.3E VKKLL MVPLFPLPLPAAQPAMAHHHHHH 253 250 8.1B VKKLL HqAILYYYLN AAQPAMAHHHHHH 254

TABLE 3 Preference sequence patterns se1ected from L3 S5 sc-dsFv 1ibraryNo. Code Sequence SEQ ID NO M13-pe1B VKKLLFAIPL VVPFYAAQPA MAHHHHHH   41 2.1A VKKLLFAIPL LPAQAMPMSR MAHHHHHH * 255 2 7.5C VKKLLFAIPL YFVLVRESSSMAHHHHHH * 256 3 1.3B VKKLLFAIPL VLVVSSRTRA MAHHHHHH * 257 4 YJ3.25VKKLLFAIPL LLSRPRAVPD MAHHHHHH * 258 5 3.8A VKKLLFAIPL CVSVRSPAFAMAHHHHHH * 259 6 1.6A VKKLLFAIPL MTTLASRTHA MAHHHHHH * 260 7 1.4HVKKLLFAIPL YLSMTRSGAA MAHHHHHH * 261 8 7.8F VKKLLFAIPL WLRSSVPVDSMAHHHHHH * 262 9 7.8H VKKLLFAIPL LSSLTRDSSS MAHHHHHH * 263 10 7.5EVKKLLFAIPL GLFTIRDSFA MAHHHHHH * 264 11 7.6C VKKLLFAIPL WLGITKPVWSMAHHHHHH * 265 12 1.3F VKKLLFAIPL YTLTPRPVFS MAHHHHHH * 266 13 1.5FVKKLLFAIPL gLALSRPSFP MAHHHHHH * 267 14 14.9A VKKLLFAIPL SSFLVADQSSMAHHHHHH * 268 15 YJ3.7 VKKLLFAIPL LLGLASPRSR MAHHHHHH * 269 16 13.1EVKKLLFAIPL LTLSNRSAWS MAHHHHHH * 270 17 2.2C VKKLLFAIPL LSLYPTRSTAMAHHHHHH * 271 18 YJ3.10 VKKLLFAIPL LTTLSRPSFS MAHHHHHH * 272 19 8.1AVKKLLFAIPL YFSRPPqPSS MAHHHHHH * 273 20 6.2H VKKLLFAIPL TMSSPPRSTSMAHHHHHH * 274 21 8.1C VKKLLFAIPL YFLRISPSAS MAHHHHHH * 275 22 1.8BVKKLLFAIPL LFLRPSAARP MAHHHHHH * 276 23 1.8C VKKLLFAIPL LWSSSRPTSQMAHHHHHH * 277 24 YJ3.41 VKKLLFAIPL YLVCSRPLHA MAHHHHHH * 278 25 10.8GVKKLLFAIPL VLQRPPSPNT MAHHHHHH * 279 26 2.7C VKKLLFAIPL AMASFRPRDQMAHHHHHH * 280 27 7.10C VKKLLFAIPL SRSLAMQPLP MAHHHHHH * 281 28 1.2AVKKLLFAIPL LSSLRSSNPE MAHHHHHH * 282 29 YJ3.4 VKKLLFAIPL SILINFRASSMAHHHHHH * 283 30 1.6B VKKLLFAIPL YWRSFWEPPA MAHHHHHH * 284 31 4.8EVKKLLFAIPL YLAAPRSTVA MAHHHHHH * 285 32 6.7H VKKLLFAIPL QYSAFSMSPRMAHHHHHH * 286 33 7.9C VKKLLFAIPL YLVSSKNSYP MAHHHHHH * 287 34 YJ3.72VKKLLFAIPL GLSVSFRTSA MAHHHHHH * 288 35 4.4C VKKLLFAIPL AMLEPTRSSAMAHHHHHH * 289 36 11.1B VKKLLFAIPL SLSLHRPALA MAHHHHHH * 290 37 6.6BVKKLLFAIPL LSASARGSYA MAHHHHHH * 291 38 YJ3.26 VKKLLFAIPL YLAVTHRAYSMAHHHHHH * 292 39 YJ3.44 VKKLLFAIPL FFSLSRYSLA MAHHHHHH * 293 40 5.4BVKKLLFAIPL YLSAPRHASP MAHHHHHH * 294 41 5.2D VKKLLFAIPL WSFSRLPSSDMAHHHHHH * 295 42 12.4E VKKLLFAIPL YLSLTKPSLS MAHHHHHH * 296 43 14.1CVKKLLFAIPL SSPATEVLSP MAHHHHHH * 297 44 6.2C VKKLLFAIPL TLFLQRSSLAMAHHHHHH * 298 45 YJ3.6 VKKLLFAIPL VFTRVPHKPS MAHHHHHH * 299 46 4.1EVKKLLFAIPL AITRSSQFPS MAHHHHHH * 300 47 6.4H VKKLLFAIPL LGDLRSSPDAMAHHHHHH * 301 48 YJ3.53 VKKLLFAIPL VTTLSTRCYA MAHHHHHH * 302 49 7.7BVKKLLFAIPL FDASLEGPAM MAHHHHHH * 303 50 11.3C VKKLLFAIPL YFSSPSSRAPMAHHHHHH * 304 51 1.12A VKKLLFAIPL WFSFPFRSAA MAHHHHHH 305 52 12.1AVKKLLFAIPL YLSMSSPARS MAHHHHHH 306 53 1.12D VKKLLFAIPL SWSLCRPVCAMAHHHHHH 307 54 4.3G VKKLLFAIPL LYCWPRHSWS MAHHHHHH 308 55 YJ3.38VKKLLFAIPL IFYTTRSSLS MAHHHHHH 309 56 YJ3.45 VKKLLFAIPL IYTLRSHSMTMAHHHHHH 310 57 2.9H VKKLLFAIPL PVPSLLGSAD MAHHHHHH 311 58 9.5AVKKLLFAIPL SLSLNSRSYP MAHHHHHH 312 59 2.7H VKKLLFAIPL FSPTSQEIRHMAHHHHHH 313 60 2.2G VKKLLFAIPL YFSCPLRVAS MAHHHHHH 314 61 YJ3.81VKKLLFAIPL VLSLNRGVFA MAHHHHHH 315 62 7.4H VKKLLFAIPL SPqVLSSSPGMAHHHHHH 316 63 4.2C VKKLLFAIPL YVNAMSSPRP MAHHHHHH 317 64 13.6DVKKLLFAIPL YFTFVRSSWC MAHHHHHH 318 65 5.8D VKKLLFAIPL FDLSSDSVSPMAHHHHHH 319 66 YJ3.47 VKKLLFAIPL YILFWRNTHA MAHHHHHH 320 67 13.7AVKKLLFAIPL SCFLSRSAFS MAHHHHHH 321 68 YJ3.83 VKKLLFAIPL FFMITSKSRSMAHHHHHH 322 69 12.6C VKKLLFAIPL IVSSSRGSFA MAHHHHHH 323 70 4.10BVKKLLFAIPL AASRPLSPAA MAHHHHHH 324 71 YJ3.46 VKKLLFAIPL WLFSPLRSYSMAHHHHHH 325 72 YJ3.56 VKKLLFAIPL FLSYVRPLSA MAHHHHHH 326 73 13.5GVKKLLFAIPL FIFTPRSVHS MAHHHHHH 327 74 2.2E VKKLLFAIPL VSSIYKNSPPMAHHHHHH 328 75 5.5H VKKLLFAIPL MSDSTAPSFA MAHHHHHH 329 76 6.4BVKKLLFAIPL TLPqPRFPSP MAHHHHHH 330 77 7.10G VKKLLFAIPL SLLADSPRRPMAHHHHHH 331 78 5.3A VKKLLFAIPL FTDNSGEPSL MAHHHHHH 332 79 11.1EVKKLLFAIPL YCMPMSRTCA MAHHHHHH 333 80 11.1D VKKLLFAIPL MSRLSYHTPSMAHHHHHH 334 81 2.2F VKKLLFAIPL LSNSRVPPSS MAHHHHHH 335 82 15.7AVKKLLFAIPL FFASMRHTqA MAHHHHHH 336 83 YJ3.5 VKKLLFAIPL LLSTIKTSFSMAHHHHHH 337 84 3.3A VKKLLFAIPL FQQSSLSSVP MAHHHHHH 338 85 16.11AVKKLLFAIPL TLILSHRSSA MAHHHHHH 339 86 11.12A VKKLLFAIPL SFSRDPSFTSMAHHHHHH 340 87 9.1B VKKLLFAIPL ALSPTRHTLA MAHHHHHH 341 88 13.9AVKKLLFAIPL NILFTVRVYA MAHHHHHH 342 89 YJ3.15 VKKLLFAIPL LASLSARCHGMAHHHHHH 343 90 12.6B VKKLLFAIPL SVTLSLRASA MAHHHHHH 344 91 15.8HVKKLLFAIPL SHDPLLLSSP MAHHHHHH 345 92 YJ3.71 VKKLLFAIPL LWSLSSRGMTMAHHHHHH 346 93 YJ3.82 VKKLLFAIPL LISYCRPVSS MAHHHHHH 347 94 9.1DVKKLLFAIPL HSVELPASPA MAHHHHHH 348 95 9.6A VKKLLFAIPL LLSTSRSSSGMAHHHHHH 349 96 YJ3.34 VKKLLFAIPL WFSCSRFALS MAHHHHHH 350 97 YJ3.28VKKLLFAIPL VCTLSSRAFS MAHHHHHH 351 98 11.1H VKKLLFAIPL YSPLARNPFSMAHHHHHH 352 99 16.9D VKKLLFAIPL FFAFSRQSSG MAHHHHHH 353 100 YJ3.70VKKLLFAIPL TFSIFSRALA MAHHHHHH 354 101 YJ3.55 VKKLLFAIPL SLFFSARAIAMAHHHHHH 355 102 9.7A VKKLLFAIPL SQPSLCDPVP MAHHHHHH 356 103 10.11AVKKLLFAIPL LASYHRVAFA MAHHHHHH 357 104 10.1F VKKLLFAIPL WQLWQLPSRPMAHHHHHH 358 105 16.8A VKKLLFAIPL FTPMYRPTSP MAHHHHHH 359 106 YJ3.27VKKLLFAIPL LLSLHRFSFA MAHHHHHH 360 107 9.5H VKKLLFAIPL SYSHPQNALAMAHHHHHH 361 108 10.12D VKKLLFAIPL YVLRSDASWG MAHHHHHH 362 109 4.2DVKKLLFAIPL FSGPPFDRTS MAHHHHHH 363 110 YJ3.66 VKKLLFAIPL FCALSRFTHAMAHHHHHH 364 111 YJ3.24 VKKLLFAIPL FSLSRPVPPL MAHHHHHH 365 112 10.7DVKKLLFAIPL SMDSFSRPFF MAHHHHHH 366 113 15.7C VKKLLFAIPL YTIIPSRASSMAHHHHHH 367 114 15.12C VKKLLFAIPL VPSANPPPLS MAHHHHHH 368 115 15.7EVKKLLFAIPL YLIKPPEGFS MAHHHHHH 369 116 YJ3.42 VKKLLFAIPL ISTLHFRAFGMAHHHHHH 370 117 YJ3.37 VKKLLFAIPL VRVMCGHSYA MAHHHHHH 371 118 YJ3.67VKKLLFAIPL VLSLSRTFSG MAHHHHHH 372 119 YJ3.75 VKKLLFAIPL WCALSRQSMPMAHHHHHH 373 120 YJ3.86 VKKLLFAIPL YFWSLRVSWP MAHHHHHH 374 121 YJ3.33VKKLLFAIPL YILSPRLPPP MAHHHHHH 375 122 YJ3.22 VKKLLFAIPL VVAAHRFSYAMAHHHHHH 376 123 YJ3.62 VKKLLFAIPL YVHLTSKAIP MAHHHHHH 377 124 YJ3.59VKKLLFAIPL SLTLYRSGWS MAHHHHHH 378 125 YJ3.18 VKKLLFAIPL YYALSGRPVTMAHHHHHH 379 126 YJ3.79 VKKLLFAIPL MLSLMRQSAP MAHHHHHH 380

TABLE 4 Preference sequence patterns se1ected from L4 S5 sc-dsFv 1ibraryNo. Code Sequence SEQ ID NO M13-pe1B VKKLLFAIPLVVPFY AAQPAMAHHH HHH   41 1.11A VKKLLFAIPLVVPFY ARPLTRIQTP HHH * 381 2 9.3D VKKLLFAIPLVVPFYLTQLSRREPS HHH * 382 3 1.6B VKKLLFAIPLVVPFY ARSLATSPSR HHH * 383 4 14.5HVKKLLFAIPLVVPFY PARSYMLVRP HHH * 384 5 12.2A VKKLLFAIPLVVPFY SRSYMLLSRPHHH * 385 6 12.6H VKKLLFAIPLVVPFY TRSALAFFLP HHH * 386 7 YJ4.13VKKLLFAIPLVVPFY SRGFTLPRLI HHH * 387 8 YJ4.1 VKKLLFAIPLVVPFY SSAFTRPIRPHHH * 388 9 12.2E VKKLLFAIPLVVPFY TRYSHAFMLI HHH * 389 10 6.10BVKKLLFAIPLVVPFY ARPMSMFRSD HHH * 390 11 8.4D VKKLLFAIPLVVPFY ASSMSqYRQNHHH * 391 12 5.9H VKKLLFAIPLVVPFY ARSYSRPPSI HHH * 392 13 10.8AVKKLLFAIPLVVPFY ASSMSRLRPH HHH * 393 14 YJ4.3 VKKLLFAIPLVVPFY CRSLSRPMLVHHH * 394 15 4.6C VKKLLFAIPLVVPFY SRSMSLHPTA HHH * 395 16 CM11VKKLLFAIPLVVPFY TRSMTRLAPP HHH * 396 17 9.8H VKKLLFAIPLVVPFY TRAMSVSHKTHHH * 397 18 13.1F VKKLLFAIPLVVPFY LLAPKPSVKR HHH * 398 19 9.7AVKKLLFAIPLVVPFY SRPAPALSRL HHH * 399 20 15.9C VKKLLFAIPLVVPFY AKAMSARYQSHHH * 400 21 CM18 VKKLLFAIPLVVPFY FASQRSSPIR HHH * 401 22 CM24VKKLLFAIPLVVPFY CLSFTSARFq HHH * 402 23 12.1A VKKLLFAIPLVVPFY PSASSRLSPKHHH * 403 24 2.10G VKKLLFAIPLVVPFY ARSYTRVPLA HHH * 404 25 CM2VKKLLFAIPLVVPFY ARSLTFLPPR HHH * 405 26 9.4C VKKLLFAIPLVVPFY TTRVNAFMLVHHH * 406 27 11.11H VKKLLFAIPLVVPFY QAFRPVPVRN HHH * 407 28 11.8HVKKLLFAIPLVVPFY TSGMSRLRSW HHH * 408 29 1.12C VKKLLFAIPLVVPFY SRSPSQLSSRHHH * 409 30 16.12H VKKLLFAIPLVVPFY AFSLSRTSSK HHH * 410 31 3.11FVKKLLFAIPLVVPFY FHRVQQFSPA HHH * 411 32 9.2B VKKLLFAIPLVVPFY LDSMLTFRRSHHH * 412 33 CM40 VKKLLFAIPLVVPFY CRSLTSPLRM HHH * 413 34 15.5BVKKLLFAIPLVVPFY SRSASFLRPI HHH * 414 35 9.2F VKKLLFAIPLVVPFY MTFqSNSPRGHHH * 415 36 CM38 VKKLLFAIPLVVPFY CRPMTLRqPV HHH * 416 37 CM5VKKLLFAIPLVVPFY VRPMSRVIMS HHH * 417 38 CM36 VKKLLFAIPLVVPFY SYGFSRPFSKHHH * 418 39 11.9G VKKLLFAIPLVVPFY TRSCFAFMLP HHH * 419 40 6.8BVKKLLFAIPLVVPFY AFSGAFRQSQ HHH * 420 41 16.6B VKKLLFAIPLVVPFY LRAGSFSAAPHHH * 421 42 CM22 VKKLLFAIPLVVPFY SHSMAPPSRR HHH * 422 43 CM31VKKLLFAIPLVVPFY CRSGTFGNIG HHH * 423 44 11.5F VKKLLFAIPLVVPFY ARSMASTPLAHHH * 424 45 YJ4.2 VKKLLFAIPLVVPFY VYPLAPRLRD HHH * 425 46 6.10HVKKLLFAIPLVVPFY SLPWRRTPFQ HHH * 426 47 10.3D VKKLLFAIPLVVPFY MRTPPLSqRIHHH * 427 48 CM28 VKKLLFAIPLVVPFY ARSLSSYNAV HHH * 428 49 12.4DVKKLLFAIPLVVPFY VHALARKSQF HHH * 429 50 CM25 VKKLLFAIPLVVPFY SRSFSSPSITHHH 430 51 13.5A VKKLLFAIPLVVPFY CRALSKPLPP HHH 431 52 12.6CVKKLLFAIPLVVPFY CRPSAPKMLL HHH 432 53 CM16 VKKLLFAIPLVVPFY SRSMSYFqPLHHH 433 54 4.2C VKKLLFAIPLVVPFY TRSLSRSIPH HHH 434 55 16.6CVKKLLFAIPLVVPFY SQLHqSPGNP HHH 435 56 10.10A VKKLLFAIPLVVPFY TRAIARPPYTHHH 436 57 10.11G VKKLLFAIPLVVPFY ARSLSTVRFP HHH 437 58 CM8VKKLLFAIPLVVPFY TRAFSSPLSN HHH 438 59 9.6D VKKLLFAIPLVVPFY NRTPTIqRDSHHH 439 60 8.4B VKKLLFAIPLVVPFY ARAVSRTVPT HHH 440 61 8.5EVKKLLFAIPLVVPFY AqSMAVPIST HHH 441 62 13.2C VKKLLFAIPLVVPFY PqPSRGFMLIHHH 442 63 CM10 VKKLLFAIPLVVPFY TRSMVFPAKV HHH 443 64 CM26VKKLLFAIPLVVPFY SRSMTLKGPE HHH 444 65 CM17 VKKLLFAIPLVVPFY AFPFSRQPNAHHH 445 66 CM7 VKKLLFAIPLVVPFY SRALTSISGM HHH 446 67 CM6 VKKLLFAIPLVVPFYCRGMSLNVTR HHH 447 68 6.10C VKKLLFAIPLVVPFY SHWRTQRPPE HHH 448 69 CM45VKKLLFAIPLVVPFY ARSFSSPPGP HHH 449 70 13.1G VKKLLFAIPLVVPFY IFPIEASARRHHH 450 71 CM39 VKKLLFAIPLVVPFY ASSMALRPRV HHH 451 72 YJ4.74VKKLLFAIPLVVPFY SRAFSSTPAM HHH 452 73 1.7F VKKLLFAIPLVVPFY SRSMVLQGPTHHH 453 74 YJ4.28 VKKLLFAIPLVVPFY SRSMTSPPYI HHH 454 75 10.3BVKKLLFAIPLVVPFY ANRPQSTKNI HHH 455 76 YJ4.56 VKKLLFAIPLVVPFY SRALTMTPSFHHH 456 77 4.6H VKKLLFAIPLVVPFY PTRLFAFMLT HHH 457 78 14.12AVKKLLFAIPLVVPFY SRAMSPIPRQ HHH 458 79 CM29 VKKLLFAIPLVVPFY ARSMGSMWQLHHH 459 80 YJ4.42 VKKLLFAIPLVVPFY SFSMTRSSPL HHH 460 81 CM42VKKLLFAIPLVVPFY SFSFIRqPLP HHH 461 82 YJ4.33 VKKLLFAIPLVVPFY NRVPSPASQTHHH 462 83 YJ4.23 VKKLLFAIPLVVPFY SFSFSKPRFS HHH 463 84 CM27VKKLLFAIPLVVPFY ARSLTQFSSV HHH 464 85 YJ4.39 VKKLLFAIPLVVPFY ARCFSSPVALHHH 465 86 11.3B VKKLLFAIPLVVPFY GASSWWLFPS HHH 466 87 YJ4.84VKKLLFAIPLVVPFY TPPQQQALLS HHH 467 88 14.1F VKKLLFAIPLVVPFY SRGFSMAFFPHHH 468 89 CM33 VKKLLFAIPLVVPFY SLAMSRPqAS HHH 469 90 13.12CVKKLLFAIPLVVPFY TYALTTFqSV HHH 470 91 YJ4.44 VKKLLFAIPLVVPFY QHAFTRPFRVHHH 471 92 CM30 VKKLLFAIPLVVPFY SRAFSSPSGS HHH 472 93 13.11GVKKLLFAIPLVVPFY TSALARSPRV HHH 473 94 4.8B VKKLLFAIPLVVPFY CRAMSSPFRPHHH 474 95 4.2B VKKLLFAIPLVVPFY STFARSFMLT HHH 475 96 9.2DVKKLLFAIPLVVPFY FPLSSRAFML HHH * 476 97 YJ4.71 VKKLLFAIPLVVPFYSRSMSTSPIL HHH 477 98 9.6H VKKLLFAIPLVVPFY SFGLqLPqPF HHH 478 99 CM37VKKLLFAIPLVVPFY SRSMSLSSDL HHH 479 100 16.3E VKKLLFAIPLVVPFY AFPLARRPINHHH 480 101 12.1B VKKLLFAIPLVVPFY TSCRAMTLPR HHH 481 102 CM23VKKLLFAIPLVVPFY TYPFSRAGPP HHH 482 103 YJ4.47 VKKLLFAIPLVVPFY ANQQALPFQLHHH 483 104 YJ4.38 VKKLLFAIPLVVPFY GWSMSLRSHS HHH 484 105 4.11HVKKLLFAIPLVVPFY SPQVVTRKDL HHH 485 106 12.9G VKKLLFAIPLVVPFY LRNAHAMASAHHH 486 107 CM44 VKKLLFAIPLVVPFY SRSGSFNVTP HHH 487 108 11.3EVKKLLFAIPLVVPFY SRPLSRVPVF HHH 488 109 11.9F VKKLLFAIPLVVPFY SKRMPPPISqHHH 489 110 CM34 VKKLLFAIPLVVPFY TRSMSSLPSP HHH 490 111 14.11DVKKLLFAIPLVVPFY CRSSSSIFPL HHH 491 112 CM15 VKKLLFAIPLVVPFY RSAHAMSIQTHHH 492 113 10.1H VKKLLFAIPLVVPFY GYCFSARIIR HHH 493 114 9.10AVKKLLFAIPLVVPFY PHLSPLqPQq HHH 494 115 CM43 VKKLLFAIPLVVPFY SFSFSRFPGLHHH 495 116 YJ4.48 VKKLLFAIPLVVPFY SSSMSLRPQF HHH 496 117 11.11DVKKLLFAIPLVVPFY SSPRARPVPP HHH 497 118 CM46 VKKLLFAIPLVVPFY ARSLSALSPYHHH 498 119 12.5C VKKLLFAIPLVVPFY PVRqLHTNLR HHH 499 120 10.2FVKKLLFAIPLVVPFY PTTSTPYqSP HHH 500 121 CM21 VKKLLFAIPLVVPFY VNALTFLPSqHHH 501 122 CM41 VKKLLFAIPLVVPFY ARSLSSPLTL HHH 502 123 YJ4.25VKKLLFAIPLVVPFY TRPPTVGLRQ HHH 503 124 CM14 VKKLLFAIPLVVPFY TRALSPMSWqHHH 504 125 YJ4.6 VKKLLFAIPLVVPFY VFPFSRPLLR HHH 505 126 CM1VKKLLFAIPLVVPFY VPRCLSMSLG HHH 506 127 YJ4.87 VKKLLFAIPLVVPFY QQPSFHPISRHHH 507 128 CM32 VKKLLFAIPLVVPFY SKAFSSFqAS HHH 508 129 10.6HVKKLLFAIPLVVPFY GYSMSqSGLT HHH 509 130 YJ4.40 VKKLLFAIPLVVPFY AQALTTRGLAHHH 510 131 YJ4.26 VKKLLFAIPLVVPFY VKSLTRPAFL HHH 511 132 12.4FVKKLLFAIPLVVPFY AqSRLRVYPP HHH 512 133 4.5B VKKLLFAIPLVVPFY PAIGFMLLRYHHH 513 134 12.3D VKKLLFAIPLVVPFY SFGTLVRPRP HHH 514 135 CM3VKKLLFAIPLVVPFY IRRPVDPVMP HHH 515 136 YJ4.19 VKKLLFAIPLVVPFY FPLRQTHRYPHHH 516 137 13.2H VKKLLFAIPLVVPFY THSMQRPTGR HHH 517 138 10.5DVKKLLFAIPLVVPFY RHTqLSSSTS HHH 518 139 15.10D VKKLLFAIPLVVPFY SCGFSRLSKAHHH 519 140 CM35 VKKLLFAIPLVVPFY SRSFSQLPHI HHH 520 141 YJ4.43VKKLLFAIPLVVPFY SSSMSQLRPF HHH 521 142 10.2B VKKLLFAIPLVVPFY CRTTFALQSSHHH 522 143 CM19 VKKLLFAIPLVVPFY AQSMSIRHNN HHH 523 144 11.4EVKKLLFAIPLVVPFY NSRFRTTPPS HHH 524 145 CM20 VKKLLFAIPLVVPFY SVSMSRYQLSHHH 525 146 CM12 VKKLLFAIPLVVPFY SSGASRLRIL HHH 526 147 YJ4.81VKKLLFAIPLVVPFY CWSLSRPRLL HHH 527 148 10.1C VKKLLFAIPLVVPFY TSRSTKLTPSHHH 528 149 11.6D VKKLLFAIPLVVPFY SRVSVAFMLM HHH 529 150 YJ4.72VKKLLFAIPLVVPFY CLGRSMAPGP HHH 530 151 14.1A VKKLLFAIPLVVPFY FVHRRDSSSLHHH 531 152 YJ4.24 VKKLLFAIPLVVPFY SLGFSRLTSL HHH 532 153 13.2BVKKLLFAIPLVVPFY ASALSRRVPq HHH 533 154 11.6B VKKLLFAIPLVVPFY TYPASWPRLRHHH 534 155 9.2G VKKLLFAIPLVVPFY SRVSLAVTPS HHH 535 156 10.11BVKKLLFAIPLVVPFY NNPFSSLSqq HHH 536 157 11.8D VKKLLFAIPLVVPFY RPLPRPFAGNHHH 537 158 CM4 VKKLLFAIPLVVPFY GFSMTQYLPq HHH 538 159 YJ4.75VKKLLFAIPLVVPFY SSALSRSFYP HHH 539 160 YJ4.61 VKKLLFAIPLVVPFY TQQRCFAMHIHHH 540 161 YJ4.85 VKKLLFAIPLVVPFY IKHFYNSRPS HHH 541 162 YJ4.51VKKLLFAIPLVVPFY FTRLPKESSP HHH 542 163 9.6G VKKLLFAIPLVVPFY LPAQPRVTRTHHH 543 164 CM13 VKKLLFAIPLVVPFY LRSMTLNTST HHH 544 165 YJ4.35VKKLLFAIPLVVPFY PDTFSYSSQD HHH 545 166 YJ4.41 VKKLLFAIPLVVPFY FRNPQLPSSAHHH 546 167 YJ4.50 VKKLLFAIPLVVPFY FRPDRTPPSS HHH 547 168 9.8CVKKLLFAIPLVVPFY qSHTILPLPA HHH 548 169 CM9 VKKLLFAIPLVVPFY SSAFqPMVSSHHH 549 170 9.7H VKKLLFAIPLVVPFY QSRRLPILPL HHH 550 171 YJ4.31VKKLLFAIPLVVPFY GQAYLPAPQL HHH 551 172 9.11B VKKLLFAIPLVVPFY TSRPRETLFLHHH 552 173 9.3G VKKLLFAIPLVVPFY TAASVVRSRD HHH 553 174 10.5FVKKLLFAIPLVVPFY VRGAAPKFSV HHH 554 175 YJ4.14 VKKLLFAIPLVVPFY FRHQPASVSTHHH 555 176 9.8B VKKLLFAIPLVVPFY PTNAIAFFLq HHH 556 177 YJ4.59VKKLLFAIPLVVPFY LKSLRSDTPN HHH 557 178 YJ4.22 VKKLLFAIPLVVPFY IKRPLPLAPTHHH 558 179 11.11F VKKLLFAIPLVVPFY ASSSKSRFML HHH 559 180 YJ4.82VKKLLFAIPLVVPFY PWKPRLLPPQ HHH 560 181 9.1H VKKLLFAIPLVVPFY SRGFMLTLRYHHH 561 182 9.8E VKKLLFAIPLVVPFY CKARGIMPVF HHH 562 183 YJ4.17VKKLLFAIPLVVPFY ASLPRLTSQS HHH 563 184 11.2B VKKLLFAIPLVVPFY qSSAFSYMLSHHH 564 185 10.7A VKKLLFAIPLVVPFY SFSSQRFLRP HHH 565 186 9.7GVKKLLFAIPLVVPFY TSSNTSRRFP HHH 566 187 11.10B VKKLLFAIPLVVPFY NqTAATAPPRHHH 567 188 10.8G VKKLLFAIPLVVPFY GAPLSWRRSY HHH 568 189 9.10DVKKLLFAIPLVVPFY CRSVWCIPRP HHH 569 190 9.1C VKKLLFAIPLVVPFY AKACLRPLQTHHH 570 191 9.6F VKKLLFAIPLVVPFY CLASSHRHRP HHH 571 192 11.3HVKKLLFAIPLVVPFY LRADSLAPKS HHH 572 193 9.9F VKKLLFAIPLVVPFY SVPQFSGRSRHHH 573 194 YJ4.78 VKKLLFAIPLVVPFY VYPARFPAKT HHH 574 195 YJ4.21VKKLLFAIPLVVPFY NFMLRHPQTF HHH 575 196 YJ4.32 VKKLLFAIPLVVPFY YVPRFPPKSAHHH 576 197 YJ4.86 VKKLLFAIPLVVPFY LSPMSRTRYV HHH 577 198 YJ4.66VKKLLFAIPLVVPFY TYPLTKPYRP HHH 578 199 YJ4.83 VKKLLFAIPLVVPFY SSYWSHRKPPHHH 579 200 10.8C VKKLLFAIPLVVPFY SPRTFAFFLM HHH 580 201 11.1AVKKLLFAIPLVVPFY LGPGIRKKPA HHH 581 202 9.4E VKKLLFAIPLVVPFY TRLCVAKVAGHHH 582 203 11.2E VKKLLFAIPLVVPFY RSLPASGASR HHH 583 204 10.5EVKKLLFAIPLVVPFY ASPRVKSYSP HHH 584 205 9.10F VKKLLFAIPLVVPFY PSRTFAFYLVHHH 585 206 9.4H VKKLLFAIPLVVPFY qqEFAMAHHH HHH 586 207 11.8BVKKLLFAIPLVVPFY PqSSKAFFLN HHH 587 208 11.2F VKKLLFAIPLVVPFY VKALRGSYPTHHH 588 209 11.7F VKKLLFAIPLVVPFY TqPSqVRYML HHH 589 210 11.9CVKKLLFAIPLVVPFY SARGqHVRPP HHH 590 211 10.11C VKKLLFAIPLVVPFY STRCPGFFLqHHH 591 212 11.6E VKKLLFAIPLVVPFY CPSVFSRTPP HHH 592 213 11.3AVKKLLFAIPLVVPFY DASSWRHFLS HHH 593

Example 4 Production of sc-dsFv Against H5 of Influenza Virus andMicroarray Test

As described above, scFvs (8a and 12a) and their disulfide forms (ds-8aand ds-12a, respectively) to various hemagglutins (HAs) from differentserotypes of influenza virus were developed. As shown in FIG. 5, theresults indicated that selected scFv phage clones against H5 ofinfluenza virus could be introduced to sc-dsFv directly but had lowerbinding affinity as compared with original scFvs. These results alsosuggested that the binding affinity could be enhanced by sc-dsFv phagepanning procedures with the signal sequences described above.

The 8aS5 protein could be concentrated to 6 mg/ml without precipitation.The array studies suggested that 4 ng/spot of ds-8a protein could detect˜10⁷ viruses in solution by using 40 nm fluorescence beads. Inconclusion, the signal sequence derived from sc-dsFv phage productionagainst VEGF from monoclonal antibody could be applied for sc-dsFv phageproduction against hemagglutinin from natural antibody repertoire. Thebinding affinity could be enhanced by sc-dsFv phage panning proceduresto produce sc-dsFv with high binding capacity and better stability thanscFv for further applications.

Example 5 Soluble Non-Fusion sc-dsFv Expressed with Suppressor E. coliStrain

The signal sequences resulting in the successful expression of thedisplayed sc-dsFv on phage rescued from suppressor E. coli strain ER2738were more likely to result in secretion of the soluble non-fusionanti-VEGF sc-dsFv in a culture medium. Signal sequence phage library L4was selected for binding to immobilized VEGF and the VEGF-bindingenriched phage variants were amplified for the next round ofselection/amplification cycle. The selection/amplification cycle wasrepeated for four rounds. After each round of selection/amplificationcycle, a random collection of 96 phage variants were picked from theamplified phage population. These phage variants were used to infect E.coli ER2738 and the soluble sc-dsFv was expressed in the overnightcultures, which were tested for binding to immobilized VEGF with ELISA.

These random collections of phage variants were also used to infect E.coli HB2151 for the same assay to determine the sc-dsFv secretion. Theresult showed that, with ER2738 as the host, 0%, 0%, 2%, and 14% of thephage variants from 1^(st), 2^(nd), 3^(rd), and 4^(th) round ofselection/amplification cycle respectively secreted functional sc-dsFvbinding to VEGF with ELISA signal greater than OD_(450 nm)>0.6. But thistrend was not found in the experiment with E. coli strain HB2151. Thisresult indicated that signal sequence alteration could restore thesecretion of the soluble non-fusion sc-dsFv and that the search for theoptimum signal sequences could be facilitated with phage-basedselection/amplification cycles on signal sequence libraries. Thisconclusion is applicable only to the E. coli suppressor strain ER2738 asthe bacteria host for the M13 phage.

Example 6 Interface Disulfide Bond Formation in the sc-dsFv

One measurement for the folding quality of the sc-dsFv is the extent ofthe interface disulfide bond formation in the sc-dsFv. This measurementwas determined by the ratio of the sc-dsFv-VEGF binding ELISA signalafter the fXa (bovine factor Xa) treatment over that before the fXatreatment. FXa cleaves substrate sequence -IEGR- in the linker peptideconnecting the two variable domains in the sc-dsFv construct. If theinterface disulfide bond was not formed in the sc-dsFv, the cleavage ofthe linker peptide would result in dissociation of the variable domainsand abolishment of the affinity against VEGF. Hence the ratio reflectsthe percentage of interface disulfide bond formation in the sc-dsFv.This measurement was validated with the positive control (anti-VEGFscFv(fXa+)/M13pIII-pelB with -IEGR- (SEQ ID NO:599) in the linkerpeptide but without the interface disulfide bond) and the negativecontrol (anti-VEGF scFv(fXa−)/M13pIII-pelB without both the fXa cuttingsite and the interface disulfide bond).

FIG. 6A compared the extent of the interface disulfide bond formation inthe secreted soluble sc-dsFv with the disulfide bond formation in thesc-dsFv displayed on phage surface for the signal sequence variants fromthe L4 library. Strong correlation between the two measurement isevident (R²=0.508, p-value=0.000158). As shown in FIG. 6A, signalsequence optimization could improve the disulfide bond formation in thesc-dsFv from ˜0% up to 40% of the secreted sc-dsFv molecule.

Another folding quality of the sc-dsFv was determined by the ratio ofthe normalized sc-dsFv-VEGF binding ELISA signal over the normalizedquantity of the secreted sc-dsFv determined by electrophoresis andWestern blot analysis. FIG. 6B compared the extent of the interfacedisulfide bond formation in the secreted soluble sc-dsFv with thefolding qualities derived from electrophoresis and ELISA measurementsfor the signal sequence variants from the L4 library. The positivecorrelation (R²=0.296, p-value=0.062) shown in FIG. 6B indicated thatthe interface disulfide bond formation enhanced the affinity for thesc-dsFv-VEGF interaction. The plot also indicated that the selectedvariants resulted in secreted sc-dsFv with up to more than 10-foldVEGF-binding signals per unit quantity of secreted sc-dsFv compared withthe positive control scFv(fXa+)/M13pIII-pelB, indicating that thesecreted sc-dsFv from these signal sequence variants folded intoantibody-like structure substantially more effectively that the scFvconstruct. This is most likely due to the stabilizing interfacedisulfide bond that is formed in the sc-dsFv but is absent in the scFvconstruct.

Example 7 Correlation Between the Stability of sc-dsFv and the Extent ofthe Interface Disulfide Bond Formation in the sc-dsFv

The effect of interface disulfide bond in stabilizing the sc-dsFvstructure was demonstrated in FIG. 7. Secreted sc-dsFv fromrepresentative variants selected from each of the three libraries wereexpressed and incubated at 37° C. for 12 days and the affinities of thesc-dsFv's against VEGF were measured along the course of incubation.FIG. 7A shows the VEGF-binding affinity plotted against the time courseof incubation for each of the selected variants. The VEGF affinity forthe control anti-VEGF scFv dropped rapidly in the first few days ofincubation, while a few variants from L4 library resulted in stablesecreted sc-dsFv that were even gaining affinities against VEGF comparedwith freshly prepared secreted protein, presumably due to theincreasingly stabilized sc-dsFv with the formation of the interfacedisulfide bond. The correlation between the two measurements shown inFIG. 7B is strong (R²=0.867 p-value=0.023), indicating that theinterface disulfide bond could be one of the most important factors instabilizing the secreted sc-dsFv in the culture medium.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features. From the above description, one skilled in the art caneasily ascertain the essential characteristics of the present invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe scope of the following claims.

What is claimed is:
 1. An isolated nucleic acid, comprising: a firstnucleotide sequence encoding a signal peptide, and a second nucleotidesequence encoding a single chain antibody capable of forming aninterface disulfide bond, the second nucleotide sequence being located3′ downstream to the first nucleotide, wherein the signal peptide hasthe amino acid sequence of VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH(SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S,T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y;X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ isA, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or Y.
 2. The nucleic acid of claim 1, further comprising athird nucleotide encoding a phage coat protein, the third nucleotidesequence being located 3′ downstream to the second nucleotide sequence.3. The nucleic acid of claim 1, wherein the nucleic acid is anexpression vector for expression a fusion protein containing the signalpeptide and the single chain antibody.
 4. The nucleic acid of claim 1,wherein the single chain antibody contains a first variable region, asecond variable region, and a protein linker connecting the first andthe second variable region, wherein the first and the second variableregion are stabilized by an interface disulfide bond.
 5. The nucleicacid library of claim 4, wherein the first variable region is a heavychain variable region (V_(H)) or a light chain variable region (V_(L)).6. The nucleic acid library of claim 4, wherein the second variableregion is a heavy chain variable region (V_(H)) or a light chainvariable region (V_(L)).
 7. The nucleic acid of claim 3, wherein theexpression vector is a phagemid.
 8. A host cell containing the nucleicacid of claim
 4. 9. A method for producing a disulfide-stabilized singlechain antibody, comprising providing a host cell containing anexpression construct, and culturing the host cell in a medium underconditions allowing expression of the disulfide-stabilized single chainantibody, wherein the expression construct includes a first nucleotidesequence encoding a signal peptide, and a second nucleotide sequenceencoding a single chain antibody capable of forming an interfacedisulfide bond, the second nucleotide sequence being located 3′downstream to the first nucleotide, and wherein the signal peptide hasthe amino acid sequence of VKKLLFAIPLVVPFYX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀HHHGH(SEQ ID NO:598), in which X₁ is A, C, D, F, G, I, L, M, N, P, Q, R, S,T, V, or Y; X₂ is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y;X₃ is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₄ isA, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X₅ is A, C, E,F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X₆ is A, C, D, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X₇ is A, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, or Y; X₈ is A, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, or Y; X₉ is A, D, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, or Y; and X₁₀ is A, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, or Y.
 10. The method of claim 9, wherein the single chainantibody contains a first variable region, a second variable region, anda protein linker connecting the first and the second variable region,wherein the first and the second variable region are stabilized by aninterface disulfide bond.
 11. The method of claim 10, wherein the firstvariable region is a heavy chain variable region (V_(H)) or a lightchain variable region (V_(L)).
 12. The method of claim 10, wherein thesecond variable region is a heavy chain variable region (V_(H)) or alight chain variable region (V_(L)).
 13. The method of claim 9, furthercomprising, after the culturing step, collecting the medium forisolating the disulfide-stabilized single chain antibody.
 14. The methodof claim 9, wherein the expression construct is a phagemid that furtherincludes a third nucleotide encoding a phage envelope protein, the thirdnucleotide sequence being located 3′ downstream to the second nucleotidesequence.
 15. The method of claim 14, further comprising, after theculturing step, collecting the medium for isolating phage particles thatdisplay the disulfide-stabilized single chain antibody.